Compositions and methods comprising serine protease variants

ABSTRACT

The present invention provides serine protease variants, more specifically subtilisin variants produced there from. Specifically, the present invention provides serine protease variants, more specifically subtilisin variants having one or more substitutions as compared to a reference serine protease. In addition, the present invention provides compositions comprising these serine protease variants, more specifically subtilisin variants. In some embodiments, the present invention provides cleaning compositions comprising at least one of these serine protease variants, more specifically subtilisin variants.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a U.S. National Stage Application ofInternational Application No. PCT/US2012/036608, filed May 4, 2012,which claims the benefit of U.S. Provisional Application Ser. No.61/482,938, filed May 5, 2011, which are hereby incorporated byreference in their entireties.

SEQUENCE LISTING

The sequence listing submitted via EFS, in compliance with 37 C.F.R.§1.52(e), is incorporated herein by reference. The sequence listing textfile submitted via EFS contains the file “40015US-SEQ-LIST.txt” createdon Sep. 25, 2013, which is 12,288 bytes in size.

FIELD OF THE INVENTION

The present invention provides serine protease variants. Specifically,the present invention provides serine protease variants having one ormore substitutions as compared to a reference serine protease. Inaddition, the present invention provides compositions comprising theseserine protease variants. In some embodiments, the present inventionprovides cleaning compositions comprising at least one of these serineprotease variants, more specifically subtilisin variants.

BACKGROUND OF THE INVENTION

Although serine proteases have long been known in the art of industrialenzymes, there remains a need for engineered proteases that are suitablefor particular conditions and uses.

SUMMARY OF THE INVENTION

In some embodiments, the present invention includes an isolatedsubtilisin variant, wherein said subtilisin variant is a mature formhaving proteolytic activity and comprises an amino acid sequencecomprising a combination of amino acid substitutions at positionsselected from Lists 1-19, and wherein amino acid positions of thesubtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

In some embodiments, the present invention includes an isolatedsubtilisin variant of Bacillus lentus subtilisin GG36 protease, whereinsaid Bacillus lentus subtilisin GG36 protease has the amino acidsequence shown in SEQ ID NO:2, and wherein said subtilisin variant hasat least 80% amino acid sequence identity to SEQ ID NO:2 and hasproteolytic activity and comprises an amino acid sequence comprising acombination of amino acid substitutions selected from Lists 1-19, andwherein amino acid positions of the subtilisin variant are numberedaccording to the numbering of corresponding amino acid positions in theamino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ shownin SEQ ID NO:1.

In some embodiments, the present invention includes nucleic acidsencoding an isolated subtilisin variant, wherein said subtilisin variantis a mature form having proteolytic activity and comprises an amino acidsequence comprising a combination of amino acid substitutions atpositions selected from Lists 1-19, and wherein amino acid positions ofthe subtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1. In someembodiments, the present invention is an expression vector, host cell ormethod for producing any of the aforementioned variants.

In some embodiments, the present invention includes nucleic acidsencoding an isolated subtilisin variant of Bacillus lentus subtilisinGG36 protease, wherein said Bacillus lentus subtilisin GG36 protease hasthe amino acid sequence shown in SEQ ID NO:2, and wherein saidsubtilisin variant has at least 80% amino acid sequence identity to SEQID NO:2 and has proteolytic activity and comprises an amino acidsequence comprising a combination of amino acid substitutions selectedfrom Lists 1-19, and wherein amino acid positions of the subtilisinvariant are numbered according to the numbering of corresponding aminoacid positions in the amino acid sequence of Bacillus amyloliquefacienssubtilisin BPN′ shown in SEQ ID NO:1. In some embodiments, the presentinvention is an expression vector, host cell or method for producing anyof the aforementioned variants.

In each embodiment listed above, the variant can have improved cleaningcompared to the GG36 protease having the amino acid sequence shown inSEQ ID NO:2.

In some embodiments, the present invention includes any of the abovevariants or nucleic acids encoding the variants, wherein the total netcharge of the variant is 0, +1, +2, +3, +4, +5, −1, −2, −3, −4, or −5relative to the total net charge of the Bacillus lentus subtilisin GG36protease.

In some embodiments, the present invention includes compositions havingat least one of the variants listed above, wherein said composition isnot a fabric and home care product. In some embodiments, the presentinvention is a method of cleaning, using the aforementioned composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides an alignment of the mature reference proteasesincluding: BPN′ (SEQ ID NO:1) and GG36 (SEQ ID NO:2). Each amino acidposition of each protease variant, more specifically subtilisin variantsdescribed herein, including each cold water protease variant, isnumbered according to the numbering of the corresponding amino acidposition in the amino acid sequence of Bacillus amyloliquefacienssubtilisin protease BPN′ (SEQ ID NO:1), as shown in FIG. 1, asdetermined by alignment of the protease variant amino acid sequence withthe Bacillus amyloliquefaciens subtilisin protease BPN′ amino acidsequence. Thus, unless otherwise specified herein, substitutionpositions are given in relationship to BPN′.

DESCRIPTION OF THE INVENTION

The present invention provides serine protease variants, morespecifically subtilisin variants. Specifically, the present inventionprovides serine protease variants, more specifically subtilisin variantshaving one or more substitutions as compared to a reference serineprotease. In addition, the present invention provides compositionscomprising these serine protease variants, more specifically subtilisinvariants. In some embodiments, the present invention provides cleaningcompositions comprising at least one of these serine protease variants,more specifically subtilisin variants.

Definitions

Unless otherwise indicated, the practice of the present inventioninvolves conventional techniques commonly used in molecular biology,protein engineering, microbiology, and recombinant DNA, which are withinthe skill of the art. Such techniques are known to those of skill in theart and are described in numerous texts and reference works well knownto those of skill in the art. All patents, patent applications, articlesand publications mentioned herein, both supra and infra, are herebyexpressly incorporated herein by reference.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. Many technicaldictionaries are known to those of skill in the art. Although anymethods and materials similar or equivalent to those described hereinfind use in the practice of the present invention, some suitable methodsand materials are described herein. Accordingly, the terms definedimmediately below are more fully described by reference to theSpecification as a whole. Also, as used herein, the singular “a”, “an”and “the” includes the plural reference unless the context clearlyindicates otherwise. Numeric ranges are inclusive of the numbersdefining the range. Unless otherwise indicated, nucleic acids arewritten left to right in 5′ to 3′ orientation; amino acid sequences arewritten left to right in amino to carboxy orientation, respectively. Itis to be understood that this invention is not limited to the particularmethodology, protocols, and reagents described, as these may vary,depending upon the context they are used by those of skill in the art.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of protein purification, molecularbiology, microbiology, recombinant DNA techniques and proteinsequencing, all of which are within the skill of those in the art.

Furthermore, the headings provided herein are not limitations of thevarious aspects or embodiments of the invention which can be had byreference to the specification as a whole. Accordingly, the termsdefined immediately below are more fully defined by reference to thespecification as a whole. Nonetheless, in order to facilitateunderstanding of the invention, a number of terms are defined below.

As used herein, the terms “protease” and “proteinase” refer to an enzymeprotein that has the ability to break down other proteins. A proteasehas the ability to conduct “proteolysis,” which begins proteincatabolism by hydrolysis of peptide bonds that link amino acids togetherin a peptide or polypeptide chain forming the protein. This activity ofa protease as a protein-digesting enzyme is referred to as “proteolyticactivity.” Many well known procedures exist for measuring proteolyticactivity (See e.g., Kalisz, “Microbial Proteinases,” In: Fiechter (ed.),Advances in Biochemical Engineering/Biotechnology, (1988)). For example,proteolytic activity may be ascertained by comparative assays whichanalyze the respective protease's ability to hydrolyze a commercialsubstrate. Exemplary substrates useful in the analysis of protease orproteolytic activity, include, but are not limited to, di-methyl casein(Sigma C-9801), bovine collagen (Sigma C-9879), bovine elastin (SigmaE-1625), and bovine keratin (ICN Biomedical 902111). Colorimetric assaysutilizing these substrates are well known in the art (See e.g., WO99/34011 and U.S. Pat. No. 6,376,450, both of which are incorporatedherein by reference). The pNA assay (See e.g., Del Mar et al., Anal.Biochem. 99:316-320 [1979]) also finds use in determining the activeenzyme concentration for fractions collected during gradient elution.This assay measures the rate at which p-nitroaniline is released as theenzyme hydrolyzes the soluble synthetic substrate,succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide(suc-AAPF-pNA). The rate of production of yellow color from thehydrolysis reaction is measured at 410 nm on a spectrophotometer and isproportional to the active enzyme concentration. In addition, absorbancemeasurements at 280 nanometers (nm) can be used to determine the totalprotein concentration. The active enzyme/total protein ratio gives theenzyme purity.

As used herein, the term “subtilisin” refers any member of the S8 serineprotease family as described in MEROPS—The Peptidase Data base (See,Rawlings et al., MEROPS: the peptidase database, Nucl Acids Res, 34Database issue, D270-272 [2006]). As described therein, the peptidasefamily S8 contains the serine endopeptidase subtilisin and itshomologues (Rawlings and Barrett, Biochem J., 290:205-218, [1993]).Family S8, also known as the subtilase family, is the second largestfamily of serine peptidases. The tertiary structures for several membersof family S8 have now been determined. A typical S8 protein structureconsists of three layers with a seven-stranded 13 sheet sandwichedbetween two layers of helices. Subtilisin (S08.001) is the typestructure for clan SB (SB). Despite the different structure, the activesites of subtilisin and chymotrypsin (S01.001) can be superimposed,which suggests the similarity is the result of convergent rather thandivergent evolution.

As used herein, the terms “protease variant,” “variant protease,”“variant serine protease,” “serine protease variant”, “subtilisinvariant”, “mutant protease,” are used in reference to proteases that aresimilar to a reference protease (which may be a wild-type subtilisinprotease), particularly in their function, but have mutations in theiramino acid sequence that make them different in sequence from thewild-type protease or any starting reference protease (i.e., “parent”protease) from which the variant protease is derived. In someembodiments, the present invention provides “GG36 variants,” (or “GG36subtilisin variants”) wherein the mutations are present in the matureGG36 sequence set forth in SEQ ID NO:2. However, it is not intended thatthe reference protease be limited to any particular amino acid sequence.In addition, it is intended that the term encompass variants of a parentprotease wherein the parent protease's sequence is at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to the amino acid sequence ofSEQ ID NO:2.

As used herein, the term “cold water protease variant” means a proteasevariant, more specifically subtilisin variants of a parent protease,wherein the B. lentus subtilisin GG36 protease has the amino acidsequence of SEQ ID NO:2, wherein said protease variant, morespecifically subtilisin variants has one or more of the followingcharacteristics: a) a Test Method 2 performance index of at least 1.1,at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, atleast 1.7, at least 1.8, at least 1.9, at least 2, from 1.1 to about 10,from 1.1 to about 8, or even from 1.1 to about 5; b) a Test Method 3performance index of at least 1.1, at least 1.2, at least 1.3, at least1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least1.9, at least 2, from 1.1 to about 10, from 1.1 to about 8, or even from1.1 to about 5; c) a Test Method 4 performance index of at least 1.0, atleast 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, atleast 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from1.0 to about 10, from 1.0 to about 8, or even from 1.0 to about 5;and/or d) a Test Method 6 performance index of at least 1.1, at least1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least1.7, at least 1.8, at least 1.9, at least 2, from 1.0 to about 10, from1.0 to about 8, or even from 1.0 to about 5; and/or e) Test Method 7performance index of at least 1.1, at least 1.2, at least 1.3, at least1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least1.9, at least 2, from 1.1 to about 15, from 1.1 to about 10, or evenfrom 1.1 to about 7. Test Method 2, Test Method 3, Test Method 4, TestMethod 6, and Test Method 7 are explicitly described infra in thesection of Example 1 entitled “Test Methods”. In addition, it isintended that the term encompass variants of a parent protease whereinthe parent protease's sequence is at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% identical to the amino acid sequence of SEQ ID NO:2.

In some embodiments of the present invention, the parent protease (i.e.,“reference” or “starting” protease) is a commercially availableprotease, including but not limited to the proteases sold under thetradenames SAVINASE®, POLARZYME®, KANNASE®, LIQUINASE®, LIQUINASEULTRA®, SAVINASE ULTRA®, OVOZYME®, (by Novozymes A/S); MAXACAL®,PROPERASE®, PURAFECT®, FN3®, FN4® and PURAFECT OXP®, PURAFAST™,PURAFECT® PRIME, PURAMAX® (by Danisco US, Genencor Division); and thoseavailable from Henkel/Kemira, namely BLAP (sequence shown in FIG. 29 ofU.S. Pat. No. 5,352,604 with the following mutationsS99D+S101R+S103A+V104I+G159S, hereinafter referred to as BLAP) and BLAPX (BLAP with S3T+V4I+V205I).

As used herein, the term “variant polypeptide” refers to a polypeptidecomprising an amino acid sequence that differs in at least one aminoacid residue from the amino acid sequence of a parent or referencepolypeptide (including but not limited to wild-type polypeptides).

As used herein, “the genus Bacillus” includes all species within thegenus “Bacillus,” as known to those of skill in the art, including butnot limited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B.stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii,B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, andB. thuringiensis. It is recognized that the genus Bacillus continues toundergo taxonomical reorganization. Thus, it is intended that the genusinclude species that have been reclassified, including but not limitedto such organisms as B. stearothermophilus, which is now named“Geobacillus stearothermophilus.” The production of resistant endosporesin the presence of oxygen is considered the defining feature of thegenus Bacillus, although this characteristic also applies to therecently named Alicyclobacillus, Arnphibacillus, Aneurinibacillus,Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus,Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus,and Virgibacillus.

The terms “polynucleotide” and “nucleic acid,” which are usedinterchangeably herein, refer to a polymer of any length of nucleotidemonomers covalently bonded in a chain. DNA (deoxyribonucleic acid), apolynucleotide comprising deoxyribonucleotides, and RNA (ribonucleicacid), a polymer of ribonucleotides, are examples of polynucleotides ornucleic acids having distinct biological function. Polynucleotides ornucleic acids include, but are not limited to, a single-, double- ortriple-stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrid, or apolymer comprising purine and pyrimidine bases, or other natural,chemically, biochemically modified, non-natural or derivatizednucleotide bases. The following are non-limiting examples ofpolynucleotides: genes, gene fragments, chromosomal fragments, expressedsequence tag(s) (EST(s)), exons, introns, messenger RNA (mRNA), transferRNA (tRNA), ribosomal RNA (rRNA), ribozymes, complementary DNA (cDNA),recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. In some embodiments, polynucleotidescomprise modified nucleotides, such as methylated nucleotides andnucleotide analogs, uracyl, other sugars and linking groups such asfluororibose and thioate, and nucleotide branches. In a particularembodiment, a sequence of nucleotides is interrupted by non-nucleotidecomponents.

As used herein, the term “mutation” refers to changes made in a startingamino acid or nucleic acid sequence. It is intended that the termencompass substitutions, insertions and deletions.

As used herein, the term “vector” refers to a nucleic acid construct orpolynucleotide construct used to introduce or transfer nucleic acid(s)or polynucleotide(s) into a target cell or tissue. A vector is typicallyused to introduce foreign DNA into another cell or tissue. A vectorgenerally comprises a DNA sequence that is a transgene and a largerpolynucleotide sequence that serves as the “backbone” of the vector. Thevector typically serves to transfers genetic information, such as theinserted transgene, to a target cell or tissue so as to isolate,multiply, or express the insert in the target cell or tissue. Vectorsinclude plasmids, cloning vectors, bacteriophages, viruses (e.g., viralvector), cosmids, expression vectors, shuttle vectors, cassettes, andthe like. A vector typically includes an origin of replication, amulticloning site, and a selectable marker. The process of inserting avector into a target cell is typically referred to as transfection. Thetransfection of a cell with a viral vector is typically referred to astransduction. The present invention includes, in some embodiments, avector that comprises a DNA sequence encoding a variant protease (e.g.,precursor or mature variant protease) that is operably linked to asuitable prosequence (e.g., secretory, signal peptide sequence, etc.)capable of effecting the expression of the DNA sequence in a suitablehost.

As used herein, the term “expression cassette,” “expression plasmid” or“expression vector” refers to a nucleic acid construct or vectorgenerated recombinantly or synthetically for the expression of a nucleicacid of interest (e.g., a foreign nucleic acid or transgene) in a targetcell. The nucleic acid of interest typically expresses a protein ofinterest. An expression vector or expression cassette typicallycomprises a promoter nucleotide sequence that drives or promotesexpression of the foreign nucleic acid. The expression vector orcassette also typically includes any other specified nucleic acidelements that permit transcription of a particular nucleic acid in atarget cell. A recombinant expression cassette can be incorporated intoa plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleicacid fragment. Some expression vectors have the ability to incorporateand express heterologous DNA fragments in a host cell. Many prokaryoticand eukaryotic expression vectors are commercially available. Selectionof appropriate expression vectors is within the knowledge of those ofskill in the art. Selection of appropriate expression vectors forexpression of a protein from a nucleic acid sequence incorporated intothe expression vector is within the knowledge of those of skill in theart.

A DNA construct is an artificially constructed segment of nucleic acidthat may be introduced into a target cell or tissue. A DNA constructtypically comprises a DNA insert comprising a nucleotide sequenceencoding a protein of interest that has been subcloned into a vector.The vector may contain bacterial resistance genes for growth in bacteriaand a promoter for expression of the protein of interest in an organism.The DNA may be generated in vitro by PCR or any other suitabletechnique(s) known to those in the art. In some embodiments, the DNAconstruct comprises a nucleic acid sequence of interest. In someembodiments, the sequence is operably linked to additional elements suchas control elements (e.g., promoters, etc.). The DNA construct mayfurther comprise a selectable marker and may further comprise anincoming sequence flanked by homology boxes. The construct may compriseother non-homologous sequences, added to the ends (e.g., stuffersequences or flanks). In some embodiments, the ends of the sequence areclosed such that the DNA construct forms a closed circle. The nucleicacid sequence of interest, which is incorporated into the DNA construct,using techniques well known in the art, may be a wild-type, mutant, ormodified nucleic acid. In some embodiments, the DNA construct comprisesone or more nucleic acid sequences homologous to the host cellchromosome. In other embodiments, the DNA construct comprises one ormore non-homologous nucleotide sequences. Once the DNA construct isassembled in vitro, it may be used, for example, to: 1) insertheterologous sequences into a desired target sequence of a host cell;and/or 2) mutagenize a region of the host cell chromosome (i.e., replacean endogenous sequence with a heterologous sequence); 3) delete targetgenes; and/or 4) introduce a replicating plasmid into the host. “DNAconstruct” is used interchangeably herein with “expression cassette.”

As used herein, a “plasmid” refers to an extrachromosomal DNA moleculewhich is capable of replicating independently from the chromosomal DNA.A plasmid is double stranded (ds) and may be circular and is typicallyused as a cloning vector.

As used herein in the context of introducing a nucleic acid sequenceinto a cell, the term “introduced” refers to any method suitable fortransferring the nucleic acid sequence into the cell. Such methods forintroduction include but are not limited to protoplast fusion,transfection, transformation, electroporation, conjugation, andtransduction (See e.g., Ferrari et al., “Genetics,” in Hardwood et al.(eds.), Bacillus, Plenum Publishing Corp., pp. 57-72 [1989]).

Transformation refers to the genetic alteration of a cell which resultsfrom the uptake, genomic incorporation, and expression of geneticmaterial (e.g., DNA).

As used herein, a nucleic acid is “operably linked” with another nucleicacid sequence when it is placed into a functional relationship withanother nucleic acid sequence. For example, a promoter or enhancer isoperably linked to a nucleotide coding sequence if the promoter affectsthe transcription of the coding sequence. A ribosome binding site may beoperably linked to a coding sequence if it is positioned so as tofacilitate translation of the coding sequence. Typically, “operablylinked” DNA sequences are contiguous. However, enhancers do not have tobe contiguous. Linking is accomplished by ligation at convenientrestriction sites. If such sites do not exist, synthetic oligonucleotideadaptors or linkers may be used in accordance with conventionalpractice.

As used herein the term “gene” refers to a polynucleotide (e.g., a DNAsegment), that encodes a polypeptide and includes regions preceding andfollowing the coding regions as well as intervening sequences (introns)between individual coding segments (exons).

As used herein, “recombinant” when used with reference to a celltypically indicates that the cell has been modified by the introductionof a heterologous nucleic acid sequence or that the cell is derived froma cell so modified. For example, a recombinant cell may comprise a genenot found in identical form within the native (non-recombinant) form ofthe cell, or a recombinant cell may comprise a native gene (found in thenative form of the cell) but which has been modified and re-introducedinto the cell. A recombinant cell may comprise a nucleic acid endogenousto the cell that has been modified without removing the nucleic acidfrom the cell; such modifications include those obtained by genereplacement, site-specific mutation, and related techniques known tothose of ordinary skill in the art. Recombinant DNA technology includestechniques for the production of recombinant DNA in vitro and transferof the recombinant DNA into cells where it may be expressed orpropagated, thereby producing a recombinant polypeptide.“Recombination,” “recombining,” and “recombined” of polynucleotides ornucleic acids refer generally to the assembly or combining of two ormore nucleic acid or polynucleotide strands or fragments to generate anew polynucleotide or nucleic acid. The recombinant polynucleotide ornucleic acid is sometimes referred to as a chimera. A nucleic acid orpolypeptide is “recombinant” when it is artificial or engineered, orderived from an artificial or engineered protein or nucleic acid.

As used herein, the term nucleic acid or gene “amplification” refers toa process by which specific DNA sequences are disproportionatelyreplicated such that the amplified nucleic acid or gene becomes presentin a higher copy number than was initially present in the genome. Insome embodiments, selection of cells by growth in the presence of a drug(e.g., an inhibitor of an inhibitable enzyme) results in theamplification of either the endogenous gene encoding the gene productrequired for growth in the presence of the drug or by amplification ofexogenous (i.e., input) sequences encoding this nucleic acid or geneproduct or both.

“Amplification” is a special case of nucleic acid replication involvingtemplate specificity. It is to be contrasted with non-specific templatereplication (i.e., replication that is template-dependent but notdependent on a specific template). Template specificity is heredistinguished from fidelity of replication (i.e., synthesis of theproper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of“target” specificity. Target sequences are “targets” in the sense thatthey are sought to be sorted out from other nucleic acid. Amplificationtechniques have been designed primarily for this sorting out.

As used herein, the term “primer” refers to an oligonucleotide (apolymer of nucleotide residues), whether occurring naturally as in apurified restriction digest or produced synthetically, which is capableof acting as a point of initiation of synthesis when placed underconditions in which synthesis of a primer extension product which iscomplementary to a nucleic acid strand is induced (i.e., in the presenceof nucleotides and an inducing agent such as DNA polymerase and at asuitable temperature and pH). A primer is preferably single stranded formaximum efficiency in amplification, but may alternatively be doublestranded. If double stranded, the primer is first treated to separateits strands before being used to prepare extension products. In someembodiments, the primer is an oligodeoxyribonucleotide. The primer mustbe sufficiently long to prime the synthesis of extension products in thepresence of the inducing agent. The exact length of a primer depends ona variety of factors, including temperature, source of primer, and theuse of the method.

As used herein, the term “probe” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, recombinantly or by PCR amplification, which is typicallycapable of hybridizing to another oligonucleotide of interest. A probemay be single-stranded or double-stranded. Probes are useful in thedetection, identification and isolation of particular gene sequences. Itis contemplated that any probe used in the present invention will belabeled with any “reporter molecule,” so that it is detectable in anydetection system, including, but not limited to enzyme (e.g., ELISA, aswell as enzyme-based histochemical assays), fluorescent, radioactive,and luminescent systems. I t is not intended that the present inventionbe limited to any particular detection system or label.

As used herein, the term “target,” when used in reference to thepolymerase chain reaction, refers to the region of nucleic acid boundedby the primers used for polymerase chain reaction. Thus, the “target” issought to be sorted out from other nucleic acid sequences. A nucleotide“segment” is a region of a nucleic acid within the target nucleic acidsequence.

As used herein, the term “polymerase chain reaction” (PCR) refers to themethods of U.S. Pat. Nos. 4,683,195 4,683,202, and 4,965,188, herebyincorporated by reference, which include methods for increasing theconcentration of a segment of a target sequence in a mixture of genomicDNA without cloning or purification. This process for amplifying thetarget sequence is well known in the art.

As used herein, the term “amplification reagents” refers to thosereagents (e.g., deoxyribonucleotide triphosphates, buffer, etc.) neededfor amplification except for primers, nucleic acid template, and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.).

As used herein, the term “restriction endonuclease” or “restrictionenzyme” refers to an enzyme (e.g., bacterial enzyme) that is capable ofcutting double-stranded or single-stranded DNA at or near a specificsequence of nucleotides known as a restriction site. The nucleotidesequence comprising the restriction site is recognized and cleaved by agiven restriction endonuclease or restriction enzyme and is frequentlythe site for insertion of DNA fragments. A restriction site can beengineered into an expression vector or DNA construct.

“Homologous recombination” refers to the exchange of DNA fragmentsbetween two DNA molecules or paired chromosomes at the site of identicalor nearly identical nucleotide sequences. In some embodiments,chromosomal integration is homologous recombination.

A nucleic acid or polynucleotide is said to “encode” a polypeptide if,in its native state or when manipulated by methods known to those ofskill in the art, it can be transcribed and/or translated to produce thepolypeptide or a fragment thereof. The anti-sense strand of such anucleic acid is also said to encode the sequence.

As is known in the art, a DNA sequence can be transcribed by an RNApolymerase to produce an RNA sequence, but an RNA sequence can bereverse transcribed by reverse transcriptase to produce a DNA sequence.

“Host strain” or “host cell” refers to a suitable host for an expressionvector comprising a DNA sequence of interest. The DNA sequence ofinterest may express a protein of interest in the host strain or hostcell.

A “protein” or “polypeptide” comprises a polymeric sequence of aminoacid residues. The terms “protein” and “polypeptide” are usedinterchangeably herein. The single and 3-letter code for amino acids asdefined in conformity with the IUPAC-IUB Joint Commission on BiochemicalNomenclature (JCBN) is used through out this disclosure. The singleletter X refers to any of the twenty amino acids. It is also understoodthat a polypeptide may be coded for by more than one nucleotide sequencedue to the degeneracy of the genetic code. Mutations are named by theone letter code for the parent amino acid, followed by a three or twodigit position number and then the one letter code for the variant aminoacid. For example, mutating glycine (G) at position 87 to serine (S) isrepresented as “G087S” or “G87S”. Multiple mutations are indicated byinserting a “-” between the mutations. Mutations at positions 87 and 90are represented as either “G087S-A090Y” or “G87S-A90Y” or “G87S+A90Y” or“G087S+A090Y”. For deletions, the one letter code “Z” is used. For aninsertion relative to the parent sequence, the one letter code “Z” is onthe left side of the position number. For a deletion, the one lettercode “Z” is on the right side of the position number. For insertions,the position number is the position number before the inserted aminoacid(s), plus 0.01 for each amino acid. For example, an insertion ofthree amino acids alanine (A), serine (S) and tyrosine (Y) betweenposition 87 and 88 is shown as “Z087.01A-Z087.02S-Z087.03Y.” Thus,combining all the mutations above plus a deletion at position 100 is:“G087S-Z087.01A-Z087.02S-Z087.03Y-A090Y-A100Z.”

A “prosequence” or “propetide sequence” refers to an amino acid sequencebetween the signal peptide sequence and mature protease sequence that isnecessary for the secretion of the protease. Cleavage of the prosequenceor propeptide sequence results in a mature active protease.

The term “signal sequence” or “signal peptide” refers to a sequence ofamino acid residues that may participate in the secretion or directtransport of the mature or precursor form of a protein. The signalsequence is typically located N-terminal to the precursor or matureprotein sequence. The signal sequence may be endogenous or exogenous.One exemplary exogenous signal sequence comprises the first seven aminoacid residues of the signal sequence from Bacillus subtilis subtilisinfused to the remainder of the signal sequence of the subtilisin fromBacillus lentus (ATCC 21536). A signal sequence is normally absent fromthe mature protein. A signal sequence is typically cleaved from theprotein by a signal peptidase after the protein is transported.

The term “hybrid signal sequence” refers to signal sequences in whichpart of sequence is obtained from the expression host fused to thesignal sequence of the gene to be expressed. In some embodiments,synthetic sequences are utilized.

The term “mature” form of a protein, polypeptide, or peptide refers tothe functional form of the protein, polypeptide, or peptide without thesignal peptide sequence and propeptide sequence.

The term “precursor” form of a protein or peptide refers to a matureform of the protein having a prosequence operably linked to the amino orcarbonyl terminus of the protein. The precursor may also have a “signal”sequence operably linked to the amino terminus of the prosequence. Theprecursor may also have additional polynucleotides that are involved inpost-translational activity (e.g., polynucleotides cleaved therefrom toleave the mature form of a protein or peptide).

The term “wild-type” in reference to an amino acid sequence or nucleicacid sequence indicates that the amino acid sequence or nucleic acidsequence is native or naturally occurring sequence. As used herein, theterm “naturally-occurring” refers to anything (e.g., proteins, aminoacids, or nucleic acid sequences) that are found in nature (i.e., havenot been manipulated by means of recombinant methods).

As used herein, the term “non-naturally occurring” refers to anythingthat is not found in nature (e.g., recombinant nucleic acids produced inthe laboratory).

As used herein with regard to amino acid residue positions,“corresponding to” or “corresponds to” or “corresponds” refers to anamino acid residue at the enumerated position in a protein or peptide,or an amino acid residue that is analogous, homologous, or equivalent toan enumerated residue in a protein or peptide. As used herein,“corresponding region” generally refers to an analogous position alongrelated proteins or a reference protein.

The terms “derived from” and “obtained from” refer to not only aprotease produced or producible by a strain of the organism in question,but also a protease encoded by a DNA sequence isolated from such strainand produced in a host organism containing such DNA sequence.Additionally, the term refers to a protease which is encoded by a DNAsequence of synthetic and/or cDNA origin and which has the identifyingcharacteristics of the protease in question. To exemplify, “proteasesderived from Bacillus” refers to those enzymes having proteolyticactivity which are naturally produced by Bacillus, as well as to serineproteases like those produced by Bacillus sources but which through theuse of genetic engineering techniques are produced by non-Bacillusorganisms transformed with a nucleic acid encoding the serine proteases.

The term “identical” in the context of two nucleic acids or polypeptidesequences refers to the residues in the two sequences that are the samewhen aligned for maximum correspondence, as measured using one of thefollowing sequence comparison or analysis algorithms.

As used herein, “homologous genes” refers to a pair of genes fromdifferent, but usually related species, which correspond to each otherand which are identical or very similar to each other. The termencompasses genes that are separated by speciation (i.e., thedevelopment of new species) (e.g., orthologous genes), as well as genesthat have been separated by genetic duplication (e.g., paralogousgenes).

As used herein, “homology” refers to sequence similarity or identity,with identity being preferred. Homology may be determined using standardtechniques known in the art (See e.g., Smith and Waterman, Adv. Appl.Math. 2:482 [1981]; Needleman and Wunsch, J. Mol. Biol. 48:443 [1970\;Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 [1988]; softwareprograms such as GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package (Genetics Computer Group, Madison, Wis.); andDevereux et al., Nucl. Acid Res. 12:387-395 [1984]). One example of auseful algorithm is PILEUP. PILEUP creates a multiple sequence alignmentfrom a group of related sequences using progressive, pair-wisealignments. It can also plot a tree showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng and Doolittle (See, Feng andDoolittle, J. Mol. Evol. 35:351-360 [1987]). The method is similar tothat described by Higgins and Sharp (See, Higgins and Sharp, CABIOS5:151-153 [1989]). Useful PILEUP parameters including a default gapweight of 3.00, a default gap length weight of 0.10, and weighted endgaps. Another example of a useful algorithm is the BLAST algorithm,described by Altschul et al., (See, Altschul et al., J. Mol. Biol.215:403-410 [1990]; and Karlin and Altschul, Proc. Natl. Acad. Sci. USA90:5873-5787 [1993]). A particularly useful BLAST program is theWU-BLAST-2 program (See, Altschul et al., Meth. Enzymol. 266:460-480[1996]). WU-BLAST-2 uses several search parameters, most of which areset to the default values. The adjustable parameters are set with thefollowing values: overlap span=1, overlap fraction=0.125, word threshold(T)=11. The HSP S and HSP S2 parameters are dynamic values and areestablished by the program itself depending upon the composition of theparticular sequence and composition of the particular database againstwhich the sequence of interest is being searched. However, the valuesmay be adjusted to increase sensitivity.

The percent sequence identity between a reference sequence and a testsequence of interest may be readily determined by one skilled in theart. The percent identity shared by polynucleotide or polypeptidesequences is determined by direct comparison of the sequence informationbetween the molecules by aligning the sequences and determining theidentity by methods known in the art. An example of an algorithm that issuitable for determining sequence similarity is the BLAST algorithm,(See, Altschul, et al., J. Mol. Biol., 215:403-410 [1990]). Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence that either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. These initial neighborhood word hitsact as starting points to find longer HSPs containing them. The wordhits are expanded in both directions along each of the two sequencesbeing compared for as far as the cumulative alignment score can beincreased. Extension of the word hits is stopped when: the cumulativealignment score falls off by the quantity X from a maximum achievedvalue; the cumulative score goes to zero or below; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLAST programuses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix(See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 [1992])alignments (B) of 50, expectation (E) of 10, M′5, N′-4, and a comparisonof both strands.

The BLAST algorithm then performs a statistical analysis of thesimilarity between two sequences (See e.g., Karlin and Altschul, supra).One measure of similarity provided by the BLAST algorithm is thesmallest sum probability (P(N)), which provides an indication of theprobability by which a match between two nucleotide or amino acidsequences would occur by chance. For example, a nucleic acid isconsidered similar to a serine protease nucleic acid of this inventionif the smallest sum probability in a comparison of the test nucleic acidto a serine protease nucleic acid is less than about 0.1, morepreferably less than about 0.01, and most preferably less than about0.001. Where the test nucleic acid encodes a serine proteasepolypeptide, it is considered similar to a specified serine proteasenucleic acid if the comparison results in a smallest sum probability ofless than about 0.5, and more preferably less than about 0.2.

Percent “identical” or “identity” in the context of two or more nucleicacid or polypeptide sequences refers to two or more sequences that arethe same or have a specified percentage of nucleic acid residues oramino acid residues, respectively, that are the same, when compared andaligned for maximum similarity, as determined using a sequencecomparison algorithm or by visual inspection. “Percent sequenceidentity” or “% identity” or “% sequence identity or “% amino acidsequence identity” of a subject amino acid sequence to a reference(i.e., query) amino acid sequence means that the subject amino acidsequence is identical (i.e., on an amino acid-by-amino acid basis) by aspecified percentage to the query amino acid sequence over a comparisonlength when the sequences are optimally aligned. Thus, 80% amino acidsequence identity or 80% identity with respect to two amino acidsequences means that 80% of the amino acid residues in two optimallyaligned amino acid sequences are identical.

“Percent sequence identity” or “% identity” or “% sequence identity or“% nucleotide sequence identity” of a subject nucleic acid sequence to areference (i.e. query) nucleic acid sequence means that the subjectnucleic acid sequence is identical (i.e., on a nucleotide-by-nucleotidebasis for a polynucleotide sequence) by a specified percentage to thequery sequence over a comparison length when the sequences are optimallyaligned. Thus, 80% nucleotide sequence identity or 80% identity withrespect to two nucleic acid sequences means that 80% of the nucleotideresidues in two optimally aligned nucleic acid sequences are identical.

In some embodiments, the “percent sequence identity” or “% sequenceidentity” or “% identity” of a subject sequence to a query sequence canbe calculated by optimally aligning the two sequences and comparing thetwo optimally aligned sequences over the comparison length. The numberof positions in the optimal alignment at which identical residues occurin both sequences is determined, thereby providing the number of matchedpositions, and the number of matched positions is then divided by thetotal number of positions of the comparison length (which, unlessotherwise specified, is the length of the query sequence). The resultingnumber is multiplied by 100 to yield the percent sequence identity ofthe subject sequence to the query sequence.

“Optimal alignment” or “optimally aligned” refers to the alignment oftwo (or more) sequences giving the highest percent identity score. Forexample, optimal alignment of two protein sequences can be achieved bymanually aligning the sequences such that the maximum number ofidentical amino acid residues in each sequence are aligned together orby using software programs or procedures described herein or known inthe art. Optimal alignment of two nucleic acid sequences can be achievedby manually aligning the sequences such that the maximum number ofidentical nucleotide residues in each sequence are aligned together orby using software programs or procedures described herein or known inthe art.

In some embodiments, two polypeptide sequences are deemed “optimallyaligned” when they are aligned using defined parameters, such as adefined amino acid substitution matrix, gap existence penalty (alsotermed gap open penalty), and gap extension penalty, so as to achievethe highest similarity score possible for that pair of sequences. TheBLOSUM62 scoring matrix (See, Henikoff and Henikoff, supra) is oftenused as a default scoring substitution matrix in polypeptide sequencealignment algorithms (e.g., BLASTP). The gap existence penalty isimposed for the introduction of a single amino acid gap in one of thealigned sequences, and the gap extension penalty is imposed for eachresidue position in the gap. Exemplary alignment parameters employedare: BLOSUM62 scoring matrix, gap existence penalty=11, and gapextension penalty=1. The alignment score is defined by the amino acidpositions of each sequence at which the alignment begins and ends (e.g.,the alignment window), and optionally by the insertion of a gap ormultiple gaps into one or both sequences, so as to achieve the highestpossible similarity score.

Optimal alignment between two or more sequences can be determinedmanually by visual inspection or by using a computer, such as, but notlimited to for example, the BLASTP program for amino acid sequences andthe BLASTN program for nucleic acid sequences (See e.g., Altschul etal., Nucleic Acids Res. 25(17):3389-3402 (1997); See also, the NationalCenter for Biotechnology Information (NCBI) website).

A polypeptide of interest may be said to be “substantially identical” toa reference polypeptide if the polypeptide of interest comprises anamino acid sequence having at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99%, or at least about 99.5% sequence identity tothe amino acid sequence of the reference polypeptide. The percentidentity between two such polypeptides can be determined manually byinspection of the two optimally aligned polypeptide sequences or byusing software programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL)using standard parameters. One indication that two polypeptides aresubstantially identical is that the first polypeptide is immunologicallycross-reactive with the second polypeptide. Typically, polypeptides thatdiffer by conservative amino acid substitutions are immunologicallycross-reactive. Thus, a polypeptide is substantially identical to asecond polypeptide, for example, where the two peptides differ only by aconservative amino acid substitution or one or more conservative aminoacid substitutions.

A nucleic acid of interest may be said to be “substantially identical”to a reference nucleic acid if the nucleic acid of interest comprises anucleotide sequence having at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99%, or at least about 99.5% sequence identity tothe nucleotide sequence of the reference nucleic acid. The percentidentity between two such nucleic acids can be determined manually byinspection of the two optimally aligned nucleic acid sequences or byusing software programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL)using standard parameters. One indication that two nucleic acidsequences are substantially identical is that the two nucleic acidmolecules hybridize to each other under stringent conditions (e.g.,within a range of medium to high stringency).

A nucleic acid or polynucleotide is “isolated” when it is partially orcompletely separated from other components, including but not limited tofor example, other proteins, nucleic acids, cells, etc. Similarly, apolypeptide, protein or peptide is “isolated” when it is partially orcompletely separated from other components, including but not limited tofor example, other proteins, nucleic acids, cells, etc. On a molarbasis, an isolated species is more abundant than are other species in acomposition. For example, an isolated species may comprise at leastabout 50%, about 70%, about 80%, about 85%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,about 99%, or about 100% (on a molar basis) of all macromolecularspecies present. Preferably, the species of interest is purified toessential homogeneity (i.e., contaminant species cannot be detected inthe composition by conventional detection methods). Purity andhomogeneity can be determined using a number of techniques well known inthe art, such as agarose or polyacrylamide gel electrophoresis of aprotein or nucleic acid sample, followed by visualization upon staining.If desired, a high-resolution technique, such as high performance liquidchromatography (HPLC) or a similar means can be utilized forpurification of the material.

The term “purified” as applied to nucleic acids or polypeptidesgenerally denotes a nucleic acid or polypeptide that is essentially freefrom other components as determined by analytical techniques well knownin the art (e.g., a purified polypeptide or polynucleotide forms adiscrete band in an electrophoretic gel, chromatographic eluate, and/ora media subjected to density gradient centrifugation). For example, anucleic acid or polypeptide that gives rise to essentially one band inan electrophoretic gel is “purified.” A purified nucleic acid orpolypeptide is at least about 50% pure, usually at least about 75%,about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%,about 99.6%, about 99.7%, about 99.8% or more pure (e.g., percent byweight on a molar basis). In a related sense, the invention providesmethods of enriching compositions for one or more molecules of theinvention, such as one or more polypeptides or polynucleotides of theinvention. A composition is enriched for a molecule when there is asubstantial increase in the concentration of the molecule afterapplication of a purification or enrichment technique. A substantiallypure polypeptide or polynucleotide of the invention (e.g., substantiallypure variant protease or polynucleotide encoding a variant protease ofthe invention, respectively) will typically comprise at least about 55%,about 60%, about 70%, about 80%, about 85%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98,about 99%, about 99.5% or more by weight (on a molar basis) of allmacromolecular species in a particular composition.

In a related sense, the invention provides methods of enrichingcompositions for one or more molecules of the invention, such as one ormore polypeptides of the invention (e.g., one or more variant proteasesof the invention) or one or more nucleic acids of the invention (e.g.,one or more nucleic acids encoding one or more variant proteases of theinvention). A composition is enriched for a molecule when there is asubstantial increase in the concentration of the molecule afterapplication of a purification or enrichment technique. A substantiallypure polypeptide or polynucleotide will typically comprise at leastabout 55%, about 60%, about 70%, about 80%, about 85%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98, about 99%, about 99.5% or more by weight (on a molar basis) ofall macromolecular species in a particular composition.

As used herein, the term “combinatorial mutagenesis” or “combinatorial”refers to methods in which libraries of nucleic acid variants of areference nucleic acid sequence are generated. In these libraries, thevariants contain one or several mutations chosen from a predefined setof mutations. The methods also provide means to introduce randommutations which were not members of the predefined set of mutations.Some such methods include those set forth in U.S. Pat. No. 6,582,914,hereby incorporated by reference. Some such combinatorial mutagenesismethods include and/or encompass methods embodied in commerciallyavailable kits (e.g., QUIKCHANGE® Multi Site-Directed Mutagenesis Kit(Stratagene), PCR fusion/extension PCR).

As used herein, “having improved properties” used in connection with avariant protease refers to a variant protease with improved or enhancedwash or cleaning performance, and/or improved or enhanced stabilityoptionally with retained wash or cleaning performance, relative to thecorresponding reference protease (e.g., wild-type or naturally-occurringprotease). The improved properties of a variant protease may compriseimproved wash or cleaning performance and/or improved stability. In someembodiments, the invention provides variant proteases of the inventionthat exhibit one of more of the following properties: improved hand washperformance, improved hand or manual dishwashing performance, improvedautomatic dishwashing performance, improved laundry performance, and/orimproved stability relative to a reference protease (e.g., wild-typeprotease, such as a wild-type subtilisin).

As used herein, the term “functional assay” refers to an assay thatprovides an indication of a protein's activity. In some embodiments, theterm refers to assay systems in which a protein is analyzed for itsability to function in its usual capacity. For example, in the case ofenzymes, a functional assay involves determining the effectiveness ofthe enzyme in catalyzing a reaction.

As used herein, the term “target property” refers to the property of thestarting gene that is to be altered. It is not intended that the presentinvention be limited to any particular target property. However, in someembodiments, the target property is the stability of a gene product(e.g., resistance to denaturation, proteolysis or other degradativefactors), while in other embodiments, the level of production in aproduction host is altered.

The term “property” or grammatical equivalents thereof in the context ofa nucleic acid, as used herein, refer to any characteristic or attributeof a nucleic acid that can be selected or detected. These propertiesinclude, but are not limited to, a property affecting binding to apolypeptide, a property conferred on a cell comprising a particularnucleic acid, a property affecting gene transcription (e.g., promoterstrength, promoter recognition, promoter regulation, enhancer function),a property affecting RNA processing (e.g., RNA splicing, RNA stability,RNA conformation, and post-transcriptional modification), a propertyaffecting translation (e.g., level, regulation, binding of mRNA toribosomal proteins, post-translational modification). For example, abinding site for a transcription factor, polymerase, regulatory factor,etc., of a nucleic acid may be altered to produce desiredcharacteristics or to identify undesirable characteristics.

The term “property” or grammatical equivalents thereof in the context ofa polypeptide (including proteins), as used herein, refer to anycharacteristic or attribute of a polypeptide that can be selected ordetected. These properties include, but are not limited to oxidativestability, substrate specificity, catalytic activity, enzymaticactivity, thermal stability, alkaline stability, pH activity profile,resistance to proteolytic degradation, K_(M), k_(cat), k_(cat)/k_(M)ratio, protein folding, inducing an immune response, ability to bind toa ligand, ability to bind to a receptor, ability to be secreted, abilityto be displayed on the surface of a cell, ability to oligomerize,ability to signal, ability to stimulate cell proliferation, ability toinhibit cell proliferation, ability to induce apoptosis, ability to bemodified by phosphorylation or glycosylation, and/or ability to treatdisease, etc.

As used herein, the term “screening” has its usual meaning in the art.In one exemplary screening process, a mutant nucleic acid or variantpolypeptide encoded therefrom is provided and a property of the mutantnucleic acid or variant polypeptide, respectively, is assessed ordetermined. The determined property of the mutant nucleic acid orvariant polypeptide may be compared to a property of the correspondingprecursor (parent) nucleic acid or to the property of the correspondingparent polypeptide, respectively.

It will be apparent to the skilled artisan that the screening procedurefor obtaining a nucleic acid or protein with an altered property dependsupon the property of the starting material the modification of which thegeneration of the mutant nucleic acid is intended to facilitate. Theskilled artisan will therefore appreciate that the invention is notlimited to any specific property to be screened for and that thefollowing description of properties lists illustrative examples only.Methods for screening for any particular property are generallydescribed in the art. For example, one can measure binding, pH,specificity, etc., before and after mutation, wherein a change indicatesan alteration. Preferably, the screens are performed in ahigh-throughput manner, including multiple samples being screenedsimultaneously, including, but not limited to assays utilizing chips,phage display, and multiple substrates and/or indicators.

As used herein, in some embodiments, a screening process encompasses oneor more selection steps in which variants of interest are enriched froma population of variants. Examples of these embodiments include theselection of variants that confer a growth advantage to the hostorganism, as well as phage display or any other method of display, wherevariants can be captured from a population of variants based on theirbinding or catalytic properties. In some embodiments, a library ofvariants is exposed to stress (e.g., heat, denaturation, etc.) andsubsequently variants that are still intact are identified in a screenor enriched by selection. It is intended that the term encompass anysuitable means for selection. Indeed, it is not intended that thepresent invention be limited to any particular method of screening.

The terms “modified nucleic acid sequence” and “modified gene” are usedinterchangeably herein to refer to a nucleic acid sequence that includesa deletion, insertion or interruption of naturally occurring (i.e.,wild-type) nucleic acid sequence. In some embodiments, the expressionproduct of the modified nucleic acid sequence is a truncated protein(e.g., if the modification is a deletion or interruption of thesequence). In some embodiments, the truncated protein retains biologicalactivity. In alternative embodiments, the expression product of themodified nucleic acid sequence is an elongated protein (e.g.,modifications comprising an insertion into the nucleic acid sequence).In some embodiments, a nucleotide insertion in the nucleic acid sequenceleads to a truncated protein (e.g., when the insertion results in theformation of a stop codon). Thus, an insertion may result in either atruncated protein or an elongated protein as an expression product.

A “mutant” nucleic acid sequence typically refers to a nucleic acidsequence that has an alteration in at least one codon occurring in ahost cell's wild-type sequence such that the expression product of themutant nucleic acid sequence is a protein with an altered amino acidsequence relative to the wild-type protein. The expression product mayhave an altered functional capacity (e.g., enhanced enzymatic activity).

As used herein, the phrase “alteration in substrate specificity” refersto changes in the substrate specificity of an enzyme. In someembodiments, a change in substrate specificity is defined as a change ink_(cat) and/or K_(m) for a particular substrate, resulting frommutations of the enzyme or alteration of reaction conditions. Thesubstrate specificity of an enzyme is determined by comparing thecatalytic efficiencies it exhibits with different substrates. Thesedeterminations find particular use in assessing the efficiency of mutantenzymes, as it is generally desired to produce variant enzymes thatexhibit greater ratios of k_(cat)/K_(m) for substrates of interest.However, it is not intended that the present invention be limited to anyparticular substrate composition or substrate specificity.

As used herein, “surface property” is used in reference to electrostaticcharge, as well as properties such as the hydrophobicity andhydrophilicity exhibited by the surface of a protein.

As used herein, the term “net charge” is defined as the sum of allcharges present in a molecule. “Net charge changes” are made to a parentprotein molecule to provide a variant that has a net charge that differsfrom that of the parent molecule (i.e., the variant has a net chargethat is not the same as that of the parent molecule). For example,substitution of a neutral amino acid with a negatively charged aminoacid or a positively charged amino acid with a neutral amino acidresults in net charge of −1 with respect to the parent molecule.Substitution of a positively charged amino acid with a negativelycharged amino acid results in a net charge of −2 with respect to theparent. Substitution of a neutral amino acid with a positively chargedamino acid or a negatively charged amino acid with a neutral amino acidresults in net charge of +1 with respect to the parent. Substitution ofa negatively charged amino acid with a positively charged amino acidresults in a net charge of +2 with respect to the parent. The net chargeof a parent protein can also be altered by deletion and/or insertion ofcharged amino acids

The terms “thermally stable” and “thermostable” and “thermostability”refer to proteases that retain a specified amount of enzymatic activityafter exposure to identified temperatures over a given period of timeunder conditions prevailing during the proteolytic, hydrolyzing,cleaning or other process of the invention, while being exposed toaltered temperatures. “Altered temperatures” encompass increased ordecreased temperatures. In some embodiments, the proteases retain atleast about 50%, about 60%, about 70%, about 75%, about 80%, about 85%,about 90%, about 92%, about 95%, about 96%, about 97%, about 98%, orabout 99% proteolytic activity after exposure to altered temperaturesover a given time period, for example, at least about 60 minutes, about120 minutes, about 180 minutes, about 240 minutes, about 300 minutes,etc.

The term “enhanced stability” in the context of an oxidation, chelator,thermal and/or pH stable protease refers to a higher retainedproteolytic activity over time as compared to other proteases (e.g.,subtilisin proteases) and/or wild-type enzymes.

The term “diminished stability” in the context of an oxidation,chelator, thermal and/or pH stable protease refers to a lower retainedproteolytic activity over time as compared to other proteases (e.g.,subtilisin proteases) and/or wild-type enzymes.

The term “cleaning activity” refers to a cleaning performance achievedby a variant protease or reference protease under conditions prevailingduring the proteolytic, hydrolyzing, cleaning, or other process of theinvention. In some embodiments, cleaning performance of a variantprotease or reference protease may be determined by using various assaysfor cleaning one or more various enzyme sensitive stains on an item orsurface (e.g., a stain resulting from food, grass, blood, ink, milk,oil, and/or egg protein). Cleaning performance of a variant or referenceprotease can be determined by subjecting the stain on the item orsurface to standard wash condition(s) and assessing the degree to whichthe stain is removed by using various chromatographic,spectrophotometric, or other quantitative methodologies. Exemplarycleaning assays and methods are known in the art and include, but arenot limited to those described in WO 99/34011 and U.S. Pat. No.6,605,458, both of which are herein incorporated by reference, as wellas those cleaning assays and methods included in the Examples providedbelow.

The term “cleaning effective amount” of a variant protease or referenceprotease refers to the amount of protease that achieves a desired levelof enzymatic activity in a specific cleaning composition. Such effectiveamounts are readily ascertained by one of ordinary skill in the art andare based on many factors, such as the particular protease used, thecleaning application, the specific composition of the cleaningcomposition, and whether a liquid or dry (e.g., granular, tablet, bar)composition is required, etc.

The term “cleaning adjunct material” refers to any liquid, solid, orgaseous material included in cleaning composition other than a variantprotease of the invention. In some embodiments, the cleaningcompositions of the present invention include one of more cleaningadjunct materials. Each cleaning adjunct material is typically selecteddepending on the particular type and form of cleaning composition (e.g.,liquid, granule, powder, bar, paste, spray, tablet, gel, foam, or othercomposition). Preferably, each cleaning adjunct material is compatiblewith the protease enzyme used in the composition.

The term “enhanced performance” in the context of cleaning activityrefers to an increased or greater cleaning activity by an enzyme oncertain enzyme sensitive stains such as egg, milk, grass, ink, oil,and/or blood, as determined by usual evaluation after a standard washcycle and/or multiple wash cycles.

The term “diminished performance” in the context of cleaning activityrefers to a decreased or lesser cleaning activity by an enzyme oncertain enzyme sensitive stains such as egg, milk, grass or blood, asdetermined by usual evaluation after a standard wash cycle.

The term “comparative performance” in the context of cleaning activityof a variant protease of the invention refers to at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, atleast about 99.5% of the cleaning activity of a comparative or referenceprotease (e.g., commercially available proteases), including, but notlimited to for example, OPTIMASE™ protease (Genencor), PURAFECT™protease products (Genencor), SAVINASE™ protease (Novozymes),BPN′-variants (See e.g., U.S. Pat. No. Re 34,606), RELASE™, DURAZYME™,EVERLASE™, KANNASE™ protease (Novozymes), MAXACAL™, MAXAPEM™, PROPERASE™proteases (Genencor; See also, U.S. Pat. No. Re 34,606, and U.S. Pat.Nos. 5,700,676; 5,955,340; 6,312,936; and 6,482,628), and B. lentusvariant protease products (e.g., those described in WO 92/21760, WO95/23221 and/or WO 97/07770). Cleaning performance can be determined bycomparing the variant proteases of the present invention with referencesubtilisin proteases in various cleaning assays concerning enzymesensitive stains such as grass, blood, ink, oil, and/or milk asdetermined by usual spectrophotometric or analytical methodologies afterstandard wash cycle conditions.

As used herein, the term “consumer product” means fabric and home careproduct. As used herein, the term “fabric and home care product” or“fabric and household care product” includes products generally intendedto be used or consumed in the form in which they are sold and that arefor treating fabrics, hard surfaces and any other surfaces, and cleaningsystems all for the care and cleaning of inanimate surfaces, as well asfabric conditioner products and other products designed specifically forthe care and maintenance of fabrics, and air care products, including:air care including air fresheners and scent delivery systems, car care,pet care, livestock care, personal care, jewelry care, dishwashing,fabric conditioning (including softening and/or freshening), laundrydetergency, laundry and rinse additive and/or care, pre-treatmentcleaning compositions, hard surface cleaning and/or treatment includingfloor and toilet bowl cleaners, glass cleaners and/or treatments, tilecleaners and/or treatments, ceramic cleaners and/or treatments, andother cleaning for consumer or institutional use. In some embodiments,the fabric and home care products are suitable for use on wounds and/orskin. “Fabric and home care product” includes consumer and institutionalproducts.

As used herein, the term “non-fabric and home care products” refers tocompositions that are added to other compositions to produce an endproduct that may be a fabric and home care product.

As used herein, the term “institutional cleaning composition” refers toproducts suitable for use in institutions including but not limited toschools, hospitals, factories, stores, corporations, buildings,restaurants, office complexes and buildings, processing and/ormanufacturing plants, veterinary hospitals, factory farms, factoryranches, etc.

As used herein, the term “cleaning and/or treatment composition” is asubset of fabric and home care products that includes, unless otherwiseindicated, compositions suitable for cleaning and/or treating items.Such products include, but are not limited to, products for treatingfabrics, hard surfaces and any other surfaces in the area of fabric andhome care, including: air care including air fresheners and scentdelivery systems, car care, dishwashing, fabric conditioning (includingsoftening and/or freshening), laundry detergency, laundry and rinseadditive and/or care, hard surface cleaning and/or treatment includingfloor and toilet bowl cleaners, granular or powder-form all-purpose or“heavy-duty” washing agents, especially cleaning detergents; liquid, gelor paste-form all-purpose washing agents, especially the so-calledheavy-duty liquid types; liquid fine-fabric detergents; hand dishwashingagents or light duty dishwashing agents, especially those of thehigh-foaming type; machine dishwashing agents, including the varioustablet, granular, liquid and rinse-aid types for household andinstitutional use: car or carpet shampoos, bathroom cleaners includingtoilet bowl cleaners; as well as cleaning auxiliaries such as bleachadditives and “stain-stick” or pre-treat types, substrate-laden productssuch as dryer added sheets.

Indeed, as used herein, “cleaning composition” or “cleaning formulation”of the invention refers to any composition of the invention useful forremoving or eliminating a compound (e.g., undesired compound) from anobject, item or surface to be cleaned, including, but not limited to forexample, a fabric, fabric item, dishware item, tableware item, glasswareitem, contact lens, other solid substrate, hair (shampoo) (includinghuman or animal hair), skin (soap or and cream), teeth (mouthwashes,toothpastes), surface of an item or object (e.g., hard surfaces, such asthe hard surface of a table, table top, wall, furniture item, floor,ceiling, non-dishware item, non-tableware item, etc.), filters,membranes (e.g., filtration membranes, including but not limited toultrafiltration membranes), etc. The term encompasses any materialand/or added compound selected for the particular type of cleaningcomposition desired and the form of the product (e.g., liquid, gel,granule, spray, or other composition), as long as the composition iscompatible with the protease and other enzyme(s) used in thecomposition. The specific selection of cleaning composition materialsare readily made by considering the surface, object, item, or fabric tobe cleaned, and the desired form of the composition for the cleaningconditions during use.

Cleaning compositions and cleaning formulations include any compositionthat is suited for cleaning, bleaching, disinfecting, and/or sterilizingany object, item, and/or surface. Such compositions and formulationsinclude, but are not limited to for example, liquid and/or solidcompositions, including cleaning or detergent compositions (e.g.,liquid, tablet, gel, bar, granule, and/or solid laundry cleaning ordetergent compositions and fine fabric detergent compositions; hardsurface cleaning compositions and formulations, such as for glass, wood,ceramic and metal counter tops and windows; carpet cleaners; ovencleaners; fabric fresheners; fabric softeners; and textile, laundrybooster cleaning or detergent compositions, laundry additive cleaningcompositions, and laundry pre-spotter cleaning compositions; dishwashingcompositions, including hand or manual dishwash compositions (e.g.,“hand” or “manual” dishwashing detergents) and automatic dishwashingcompositions (e.g., “automatic dishwashing detergents”).

Cleaning composition or cleaning formulations, as used herein, include,unless otherwise indicated, granular or powder-form all-purpose orheavy-duty washing agents, especially cleaning detergents; liquid,granular, gel, solid, tablet, or paste-form all-purpose washing agents,especially the so-called heavy-duty liquid (HDL) detergent or heavy-dutypowder detergent (HDD) types; liquid fine-fabric detergents; hand ormanual dishwashing agents, including those of the high-foaming type;hand or manual dishwashing, automatic dishwashing, or dishware ortableware washing agents, including the various tablet, powder, solid,granular, liquid, gel, and rinse-aid types for household andinstitutional use; liquid cleaning and disinfecting agents, includingantibacterial hand-wash types, cleaning bars, mouthwashes, denturecleaners, car shampoos, carpet shampoos, bathroom cleaners; hairshampoos and/or hair-rinses for humans and other animals; shower gelsand foam baths and metal cleaners; as well as cleaning auxiliaries, suchas bleach additives and “stain-stick” or pre-treat types. In someembodiments, granular compositions are in “compact” form; in someembodiments, liquid compositions are in a “concentrated” form.

As used herein, “fabric cleaning compositions” include hand and machinelaundry detergent compositions including laundry additive compositionsand compositions suitable for use in the soaking and/or pretreatment ofstained fabrics (e.g., clothes, linens, and other textile materials).

As used herein, “non-fabric cleaning compositions” include non-textile(i.e., non-fabric) surface cleaning compositions, including, but notlimited to for example, hand or manual or automatic dishwashingdetergent compositions, oral cleaning compositions, denture cleaningcompositions, and personal cleansing compositions.

As used herein, the term “fabric and/or hard surface cleaning and/ortreatment composition” is a subset of cleaning and treatmentcompositions that includes, unless otherwise indicated, granular orpowder-form all-purpose or “heavy-duty” washing agents, especiallycleaning detergents; liquid, gel or paste-form all-purpose washingagents, especially the so-called heavy-duty liquid types; liquidfine-fabric detergents; hand dishwashing agents or light dutydishwashing agents, especially those of the high-foaming type; machinedishwashing agents, including the various tablet, granular, liquid andrinse-aid types for household and institutional use; liquid cleaning anddisinfecting agents, car or carpet shampoos, bathroom cleaners includingtoilet bowl cleaners; fabric conditioning products including softeningand/or freshening that may be in liquid, solid and/or dryer sheet form;as well as cleaning auxiliaries such as bleach additives and“stain-stick” or pre-treat types, substrate-laden products such as dryeradded sheets. All of such products which are applicable may be instandard, concentrated or even highly concentrated form even to theextent that such products may in certain aspect be non-aqueous.

As used herein, the term “detergent composition” or “detergentformulation” is used in reference to a composition intended for use in awash medium for the cleaning of soiled or dirty objects, includingparticular fabric and/or non-fabric objects or items. Such compositionsof the present invention are not limited to any particular detergentcomposition or formulation. Indeed, in some embodiments, the detergentsof the invention comprise at least one variant protease of the inventionand, in addition, one or more surfactants, transferase(s), hydrolyticenzymes, oxido reductases, builders (e.g., a builder salt), bleachingagents, bleach activators, bluing agents, fluorescent dyes, cakinginhibitors, masking agents, enzyme activators, antioxidants, and/orsolubilizers. In some instances, a builder salt is a mixture of asilicate salt and a phosphate salt, preferably with more silicate (e.g.,sodium metasilicate) than phosphate (e.g., sodium tripolyphosphate).Some compositions of the invention, such as, but not limited to,cleaning compositions or detergent compositions, do not contain anyphosphate (e.g., phosphate salt or phosphate builder).

As used herein, the term “bleaching” refers to the treatment of amaterial (e.g., fabric, laundry, pulp, etc.) or surface for a sufficientlength of time and/or under appropriate pH and/or temperature conditionsto effect a brightening (i.e., whitening) and/or cleaning of thematerial. Examples of chemicals suitable for bleaching include, but arenot limited to, for example, ClO₂, H₂O₂, peracids, NO₂, etc.

As used herein, “wash performance” of a protease (e.g., a variantprotease of the invention) refers to the contribution of a variantprotease to washing that provides additional cleaning performance to thedetergent as compared to the detergent without the addition of thevariant protease to the composition. Wash performance is compared underrelevant washing conditions. In some test systems, other relevantfactors, such as detergent composition, sud concentration, waterhardness, washing mechanics, time, pH, and/or temperature, can becontrolled in such a way that condition(s) typical for householdapplication in a certain market segment (e.g., hand or manualdishwashing, automatic dishwashing, dishware cleaning, tablewarecleaning, fabric cleaning, etc.) are imitated.

The term “relevant washing conditions” is used herein to indicate theconditions, particularly washing temperature, time, washing mechanics,sud concentration, type of detergent and water hardness, actually usedin households in a hand dishwashing, automatic dishwashing, or laundrydetergent market segment.

The term “improved wash performance” is used to indicate that a betterend result is obtained in stain removal under relevant washingconditions, or that less variant protease, on weight basis, is needed toobtain the same end result relative to the corresponding wild-type orstarting parent protease.

As used herein, the term “disinfecting” refers to the removal ofcontaminants from the surfaces, as well as the inhibition or killing ofmicrobes on the surfaces of items. It is not intended that the presentinvention be limited to any particular surface, item, or contaminant(s)or microbes to be removed.

The “compact” form of the cleaning compositions herein is best reflectedby density and, in terms of composition, by the amount of inorganicfiller salt. Inorganic filler salts are conventional ingredients ofdetergent compositions in powder form. In conventional detergentcompositions, the filler salts are present in substantial amounts,typically about 17 to about 35% by weight of the total composition. Incontrast, in compact compositions, the filler salt is present in amountsnot exceeding about 15% of the total composition. In some embodiments,the filler salt is present in amounts that do not exceed about 10%, ormore preferably, about 5%, by weight of the composition. In someembodiments, the inorganic filler salts are selected from the alkali andalkaline-earth-metal salts of sulfates and chlorides. In someembodiments, the filler salt is sodium sulfate.

The position of an amino acid residue in a given amino acid sequence istypically numbered herein using the numbering of the position of thecorresponding amino acid residue of the B. amyloliquefaciens subtilisinBPN′ amino acid sequence shown in SEQ ID NO:1. The B. amyloliquefacienssubtilisin BPN′ amino acid sequence of SEQ ID NO:1, thus serves as areference sequence. A given amino acid sequence, such as a variantprotease amino acid sequence described herein, can be aligned with theBPN′ sequence (SEQ ID NO:1) using an alignment algorithm as describedherein, and an amino acid residue in the given amino acid sequence thataligns (preferably optimally aligns) with an amino acid residue in theBPN′ sequence can be conveniently numbered by reference to thecorresponding amino acid residue in the subtilisin BPN′ sequence.Alternatively, if amino acid residue positions of the subtilisin variantprotease sequences are numbered using the actual numbering of the aminoacid residue positions in the GG36 amino acid sequence (SEQ ID NO:2),and not by reference to corresponding amino acid positions in the BPN′sequence upon alignment, the subtilisin variant protease can bedescribed as a variant protease of the GG36 protease shown in SEQ IDNO:2

Generally, the nomenclature used herein and many of the laboratoryprocedures in cell culture, molecular genetics, molecular biology,nucleic acid chemistry, and protein chemistry described below are wellknown and commonly employed by those of ordinary skill in the art.Methods for production and manipulation of recombinant nucleic acidmethods, nucleic acid synthesis, cell culture methods, and transgeneincorporation (e.g., transfection, electroporation) are known to thoseskilled in the art and are described in numerous standard texts.Oligonucleotide synthesis and purification steps are typically performedaccording to specifications. Techniques and procedures are generallyperformed according to conventional methods well known in the art andvarious general references that are provided throughout this document.Procedures therein are believed to be well known to those of ordinaryskill in the art and are provided for the convenience of the reader.

Polypeptides of the Invention

The present invention provides novel polypeptides, which may becollectively referred to as “polypeptides of the invention.”Polypeptides of the invention include isolated, recombinant,substantially pure, or non-naturally occurring variant proteasepolypeptides, including for example, subtilisin variant polypeptides,having enzymatic activity (e.g., proteolytic activity). In someembodiments, the polypeptides of the invention, such as a subtilisinvariant, or a variant of GG36 having the sequence of SEQ ID NO:2, hasimproved cleaning performance compared to the parent, e.g. GG36 proteasehaving sequence of SEQ ID NO:2. In some embodiments, polypeptides of theinvention are useful in cleaning applications and may be incorporatedinto cleaning compositions that are useful in methods of cleaning anitem or a surface (e.g., of surface of an item) in need of cleaning.

In some embodiments, a variant protease of the invention comprises a“variant subtilisin.” In some embodiments, the invention provides a“Bacillus sp. variant protease.” In some embodiments, the inventionprovides a “Bacillus sp. variant subtilisin.” In some embodiments, theinvention provides an isolated subtilisin variant. In some embodiments,the invention provides an isolated subtilisin variant of Bacillus lentussubtilisin GG36 protease, wherein said Bacillus lentus subtilisin GG36protease comprises the amino acid sequence shown in SEQ ID NO:2.

In any of the above embodiments, the invention includes an isolated,recombinant, substantially pure, or non-naturally occurring variantprotease having proteolytic activity, which polypeptide comprises apolypeptide sequence having at least about 80%, at least about 85%, atleast about 86%, at least about 87%, at least about 88%, at least about89%, at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, atleast about 99.5%, or 100% sequence identity to the amino acid sequencesencoding the variant proteases provided herein.

As noted above, the variant protease polypeptides of the invention haveenzymatic activities (e.g., proteolytic activities) and thus are usefulin cleaning applications, including but not limited to, methods forcleaning dishware items, tableware items, fabrics, and items having hardsurfaces (e.g., the hard surface of a table, table top, wall, furnitureitem, floor, ceiling, etc.). Exemplary cleaning compositions comprisingone or more variant protease polypeptides of the invention are describedinfra. The enzymatic activity (e.g., protease activity) of a variantprotease polypeptide of the invention can be determined readily usingprocedures well known to those of ordinary skill in the art. TheExamples presented infra describe methods for evaluating the enzymaticactivity, cleaning performance, and/or washing performance. Theperformance of variant proteases of the invention in removing stains(e.g., a proteinaceous stain), cleaning hard surfaces, or cleaninglaundry, dishware or tableware item(s) can be readily determined usingprocedures well known in the art and/or by using procedures set forth inthe Examples.

A polypeptide of the invention can be subject to various changes, suchas one or more amino acid insertions, deletions, and/or substitutions,either conservative or non-conservative, including where such changes donot substantially alter the enzymatic activity of the polypeptide.Similarly, a nucleic acid of the invention can also be subject tovarious changes, such as one or more substitutions of one or morenucleic acids in one or more codons such that a particular codon encodesthe same or a different amino acid, resulting in either a silentvariation (e.g., mutation in a nucleotide sequence results in a silentmutation in the amino acid sequence, for example when the encoded aminoacid is not altered by the nucleic acid mutation) or non-silentvariation, one or more deletions of one or more nucleic acids (orcodons) in the sequence, one or more additions or insertions of one ormore nucleic acids (or codons) in the sequence, and/or cleavage of orone or more truncations of one or more nucleic acids (or codons) in thesequence. Many such changes in the nucleic acid sequence may notsubstantially alter the enzymatic activity of the resulting encodedvariant protease compared to the variant protease encoded by theoriginal nucleic acid sequence. A nucleic acid of the invention can alsobe modified to include one or more codons that provide for optimumexpression in an expression system (e.g., bacterial expression system),while, if desired, said one or more codons still encode the same aminoacid(s).

In some embodiments, the present invention provides a genus ofpolypeptides comprising variant protease polypeptides having the desiredenzymatic activity (e.g., protease activity or cleaning performanceactivity) which comprise sequences having the amino acid substitutionsdescribed herein and also which comprise one or more additional aminoacid substitutions, such as conservative and non-conservativesubstitutions, wherein the polypeptide exhibits, maintains, orapproximately maintains the desired enzymatic activity (e.g., proteaseactivity or subtilisin activity, as reflected in the cleaning activityor performance of the variant protease). Amino acid substitutions inaccordance with the invention may include, but are not limited to, oneor more non-conservative substitutions and/or one or more conservativeamino acid substitutions. A conservative amino acid residue substitutiontypically involves exchanging a member within one functional class ofamino acid residues for a residue that belongs to the same functionalclass (identical amino acid residues are considered functionallyhomologous or conserved in calculating percent functional homology). Aconservative amino acid substitution typically involves the substitutionof an amino acid in an amino acid sequence with a functionally similaramino acid. For example, alanine, glycine, serine, and threonine arefunctionally similar and thus may serve as conservative amino acidsubstitutions for one another. Aspartic acid and glutamic acid may serveas conservative substitutions for one another. Asparagine and glutaminemay serve as conservative substitutions for one another. Arginine,lysine, and histidine may serve as conservative substitutions for oneanother. Isoleucine, leucine, methionine, and valine may serve asconservative substitutions for one another. Phenylalanine, tyrosine, andtryptophan may serve as conservative substitutions for one another.

Other conservative amino acid substitution groups can be envisioned. Forexample, amino acids can be grouped by similar function or chemicalstructure or composition (e.g., acidic, basic, aliphatic, aromatic,sulfur-containing). For instance, an aliphatic grouping may comprise:Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I). Othergroups containing amino acids that are considered conservativesubstitutions for one another include: aromatic: Phenylalanine (F),Tyrosine (Y), Tryptophan (W); sulfur-containing: Methionine (M),Cysteine (C); Basic: Arginine (R), Lysine (K), Histidine (H); Acidic:Aspartic acid (D), Glutamic acid (E); non-polar uncharged residues,Cysteine (C), Methionine (M), and Proline (P); hydrophilic unchargedresidues: Serine (S), Threonine (T), Asparagine (N), and Glutamine (Q).Additional groupings of amino acids are well-known to those of skill inthe art and described in various standard textbooks. Listing of apolypeptide sequence herein, in conjunction with the above substitutiongroups, provides an express listing of all conservatively substitutedpolypeptide sequences.

More conservative substitutions exist within the amino acid residueclasses described above, which also or alternatively can be suitable.Conservation groups for substitutions that are more conservativeinclude: valine-leucine-isoleucine, phenylalanine-tyrosine,lysine-arginine, alanine-valine, and asparagine-glutamine. Thus, forexample, in some embodiments, the invention provides an isolated orrecombinant variant protease polypeptide (e.g., variant subtilisin)having proteolytic activity, said variant protease polypeptidecomprising an amino acid sequence having at least about 90%, about 95%,about 96%, about 97%, about 98%, about 99%, or about 99.5% sequenceidentity to the amino acid sequence of SEQ ID NO:2. A conservativesubstitution of one amino acid for another in a variant protease of theinvention is not expected to alter significantly the enzymatic activityor cleaning performance activity of the variant protease. Enzymaticactivity or cleaning performance activity of the resultant protease canbe readily determined using the standard assays and the assays describedherein.

Conservatively substituted variations of a polypeptide sequence of theinvention (e.g., variant proteases of the invention) includesubstitutions of a small percentage, sometimes less than about 25%,about 20%, about 15%, about 14%, about 13%, about 12%, about 11%, about10%, about 9%, about 8%, about 7%, or about 6% of the amino acids of thepolypeptide sequence, or less than about 5%, about 4%, about 3%, about2%, or about 1%, of the amino acids of the polypeptide sequence, with aconservatively selected amino acid of the same conservative substitutiongroup.

As described elsewhere herein in greater detail and in the Examplesprovided herein, polypeptides of the invention may have cleaningabilities that may be compared to known proteases, including knownsubtilisins. Exemplary known subtilisin proteases include, but are notlimited to, for example, B. lentus subtilisin GG36, B. amyloliquefacienssubtilisin BPN′, B. amyloliquefaciens subtilisin BPN′-Y217L, and B.clausii PB92. The amino acid sequence of the mature B. lentus subtilisinGG36 protein is:

(SEQ ID NO: 2) AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR

The amino acid sequence of mature B. amyloliquefaciens subtilisin BPN′protein is:

(SEQ ID NO: 1) AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETNPFQDNNSHGTHVAGTVAALNNSIGVLGVAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGTSGSSSTVGYPGKYPSVIAVGAVDSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPNWTNTQVRSSLENTTTKLGDSFYYGKGLINVQAAAQ

The present invention provides an isolated subtilisin variant, whereinsaid subtilisin variant is a mature form having proteolytic activity andcomprises an amino acid sequence comprising a combination of amino acidsubstitutions at positions selected from T022R+V104I+Q245R,G020R+S024R+S101A, T022R+S103A+V104I, T022R+V104I+A232V,T022R+S103A+Q245R, T022R+A232V+Q245R, T022R+S103A+A232V,T022R+S103A+V104I+A232V, T022R+V104I+A232V+Q245R,T022R+S103A+V104I+Q245R, T022R+S103A+A232V+Q245R,T022R+S103A+V104I+A232V+Q245R, T022A+N076D+S103A+V104I+A232V+Q245R,T022A+N076D+S103A+V104I+A232V, T022A+N076D+V104I+A232V+Q245R,T022A+N076D+S103A+A232V+Q245R, T022A+N076D+S103A+V104I+Q245R,T022A+N076D+V104I+Q245R, T022A+N076D+A232V+Q245R,T022A+N076D+V104I+A232V, T022A+N076D+S103A+Q245R,T022A+N076D+S103A+A232V, T022A+N076D+S103A+V104I, T022A+N076D+S103A,T022A+N076D+V104I, T022A+N076D+A232V, T022A+N076D+Q245R,G020R+S101A+T213A, G020R+R045T+S101A, G020R+S101A+G211Q,S024R+S101A+T213A, G020R+T022W+S101A, S024R+S101A+G211Q,N043R+S101A+T213A, N043R+R045T+S101A, S024R+N043R+S101A,S024R+R045T+S101A, S101A+G211Q+T213A, G020R+N043R+S101A,R045T+S101A+T213A, T022W+S024R+S101A, T022R+S101G+V104I,T022W+S101A+T213A, T022R+S101G+Q245R, T022W+S101A+G211Q,G020R+S024R+Q245R, T022R+S101G+V104I+Q245R, T022R+S101G+A232V,T022R+S101G+S103A, T022R+S101G+S103A+V104I, T022R+S101G+S103A+Q245R,T022R+S101G+A232V+Q245R, T022R+S101G+V104I+A232V,G020R+S024R+S101A+T213A, G020R+S024R+T213AS101N+V104I+A232V,S101N+S103A+V104I, S101N+S103A+A232V, S101N+S103A+V104I+A232V,G020R+S103A+A232V, G020R+V104I+A232V, G020R+S103A+V104I+A232V,G020R+S103A+V104I, N018R+N076D+N116A, N018R+N076D+T213A,N018R+N076D+S101A, N018R+N076D+A215F, or N018R+N076D+G211Q (List 1), andwherein amino acid positions of the subtilisin variant are numberedaccording to the numbering of corresponding amino acid positions in theamino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ shownin SEQ ID NO:1.

The present invention further provides an isolated subtilisin variant,wherein said subtilisin variant is a mature form having proteolyticactivity and comprises an amino acid sequence comprising a combinationof amino acid substitutions at positionsT022A+N076D+S103A+V104I+A232V+Q245R (List 2), and wherein amino acidpositions of the subtilisin variant are numbered according to thenumbering of corresponding amino acid positions in the amino acidsequence of Bacillus amyloliquefaciens subtilisin BPN′ shown in SEQ IDNO:1.

The present invention further provides an isolated subtilisin variant,wherein said subtilisin variant is a mature form having proteolyticactivity and comprises an amino acid sequence comprising a combinationof amino acid substitutions at positions selected fromT022R+S103A+V104I+A232V+Q245R, T022A+N076D+S103A+V104I+A232V,T022A+N076D+V104I+A232V+Q245R, T022A+N076D+S103A+A232V+Q245R, orT022A+N076D+S103A+V104I+Q245R (List 3), and wherein amino acid positionsof the subtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention further provides an isolated subtilisin variant,wherein said subtilisin variant is a mature form having proteolyticactivity and comprises an amino acid sequence comprising a combinationof amino acid substitutions at positions selected fromT022R+S103A+V104I+A232V, T022R+V104I+A232V+Q245R,T022R+S103A+V104I+Q245R, T022R+S103A+A232V+Q245R,T022A+N076D+V104I+Q245R, T022A+N076D+A232V+Q245R,T022A+N076D+V104I+A232V, T022A+N076D+S103A+Q245R,T022A+N076D+S103A+A232V, T022A+N076D+S103A+V104I,T022R+S101G+V104I+Q245R, T022R+S101G+S103A+V104I,T022R+S101G+S103A+Q245R, T022R+S101G+A232V+Q245R,T022R+S101G+V104I+A232V, G020R+S024R+S101A+T213A,S101N+S103A+V104I+A232V or G020R+S103A+V104I+A232V (List 4), and whereinamino acid positions of the subtilisin variant are numbered according tothe numbering of corresponding amino acid positions in the amino acidsequence of Bacillus amyloliquefaciens subtilisin BPN′ shown in SEQ IDNO:1.

The present invention further provides an isolated subtilisin variant,wherein said subtilisin variant is a mature form having proteolyticactivity and comprises an amino acid sequence comprising a combinationof amino acid substitutions at positions selected fromT022R+V104I+Q245R, G020R+S024R+S101A, T022R+S103A+V104I,T022R+V104I+A232V, T022R+S103A+Q245R, T022R+A232V+Q245R,T022R+S103A+A232V, T022A+N076D+S103A, T022A+N076D+V104I,T022A+N076D+A232V, T022A+N076D+Q245R, G020R+S101A+T213A,G020R+R045T+S101A, G020R+S101A+G211Q, S024R+S101A+T213A,G020R+T022W+S101A, S024R+S101A+G211Q, N043R+S101A+1213A,N043R+R045T+S101A, S024R+N043R+S101A, S024R+R045T+S101A,S101A+G211Q+T213A, G020R+N043R+S101A, R045T+S101A+T213A,T022W+S024R+S101A, T022R+S101G+V104I, T022W+S101A+T213A,T022R+S101G+Q245R, T022W+S101A+G211Q, G020R+S024R+Q245R,T022R+S101G+A232V, T022R+S101G+S103A, G020R+S024R+T213A,S101N+V104I+A232V, S101N+S103A+V104I, S101N+S103A+A232V,G020R+S103A+A232V, G020R+V104I+A232V, G020R+S103A+V104I,N018R+N076D+N116A, N018R+N076D+T213A, N018R+N076D+S101A,N018R+N076D+A215F, or N018R+N076D+G211Q (List 5), and wherein amino acidpositions of the subtilisin variant are numbered according to thenumbering of corresponding amino acid positions in the amino acidsequence of Bacillus amyloliquefaciens subtilisin BPN′ shown in SEQ IDNO:1.

The present invention further provides an isolated subtilisin variant,wherein said subtilisin variant is a mature form having proteolyticactivity and comprises an amino acid sequence comprising a combinationof amino acid substitutions at positions selected fromS101N+V104I+A232V, S101N+S103A+V104I, S101N+S103A+A232V, orS101N+S103A+V104I+A232V (List 6), and wherein amino acid positions ofthe subtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention further provides an isolated subtilisin variant,wherein said subtilisin variant is a mature form having proteolyticactivity and comprises an amino acid sequence comprising a combinationof amino acid substitutions at positions S101N+S103A+V104I+A232V (List7), and wherein amino acid positions of the subtilisin variant arenumbered according to the numbering of corresponding amino acidpositions in the amino acid sequence of Bacillus amyloliquefacienssubtilisin BPN′ shown in SEQ ID NO:1.

The present invention further provides an isolated subtilisin variant,wherein said subtilisin variant is a mature form having proteolyticactivity and comprises an amino acid sequence comprising a combinationof amino acid substitutions at positions selected fromS101N+V104I+A232V, S101N+S103A+V104I, or S101N+S103A+A232V (List 8), andwherein amino acid positions of the subtilisin variant are numberedaccording to the numbering of corresponding amino acid positions in theamino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ shownin SEQ ID NO:1.

The present invention further provides an isolated subtilisin variant,wherein said subtilisin variant is a mature form having proteolyticactivity and comprises an amino acid sequence comprising a combinationof amino acid substitutions at positions selected fromS103A+V104I+S188D, S188D+Q245R+N248D, S103A+S188D+Q245R,V104I+S188D+Q245R, S103A+V104I+S188D+Q245R, T022A+S103A+S188D,T022A+V104I+S188D, T022A+S103A+V104I+S188D, G159D+S188D+Q245R,S103A+S188D+A232V, S103A+G159D+S188D, V104I+S188D+A232V,V104I+G159D+S188D, S103A+V104I+S188D+A232V, S103A+V104I+G159D+S188D,S103A+S188D+N248D, V104I+S188D+N248D, S103A+V104I+S188D+N248D,G159D+S188D+N248D, S188D+A232V+Q245R, S103A+S188D+A232V+Q245R,V104I+S188D+A232V+Q245R, S103A+V104I+S188D+A232V+Q245R,T022A+S188D+Q245R, T022A+S103A+S188D+Q245R, T022A+V104I+S188D+Q245R,S103A+S188D+E271F, V104I+S188D+E271F, T022A+S103A+V104I+S188D+Q245R,S103A+V104I+S188D+E271F, T022A+S188D+N248D, T022A+S103A+S188D+N248D,T022A+V104I+S188D+N248D, S103A+S188D+Q245R+N248D,T022A+S103A+V104I+S188D+N248D, V104I+S188D+Q245R+N248D,S103A+V104I+S188D+Q245R+N248D, T022A+S188D+A232V,T022A+S103A+S188D+A232V, T022A+V104I+S188D+A232V,T022A+S103A+V104I+S188D+A232V, S188D+A232V+N248D, S188D+Q245R+E271F,T022A+S188D+E271F, T022A+S188D+Q245R+N248D, S103A+S188D+A232V+N248D,S103A+S188D+Q245R+E271F, S188D+N248D+E271F, V104I+S188D+A232V+N248D,V104I+S188D+Q245R+E271F, T022A+S103A+S188D+E271F,T022A+S103A+S188D+Q245R+N248D, S103A+S188D+N248D+E271F,T022A+V104I+S188D+E271F, T022A+V104I+S188D+Q245R+N248D,S103A+V104I+S188D+Q245R+E271F, S103A+V104I+S188D+A232V+N248D,V104I+S188D+N248D+E271F, S188D+A232V+E271F,T022A+S103A+V104I+S188D+E271F, T022A+S103A+V104I+S188D+Q245R+N248D,S103A+V104I+S188D+N248D+E271F, S103A+S188D+A232V+E271F,V104I+S188D+A232V+E271F, T022A+S188D+A232V+Q245R,T022A+S188D+Q245R+E271F, T022A+S188D+N248D+E271F,S103A+V104I+S188D+A232V+E271F, T022A+S103A+S188D+A232V+Q245R,S188D+A232V+Q245R+N248D, S188D+Q245R+N248D+E271F,T022A+S188D+A232V+N248D, T022A+S103A+S188D+Q245R+E271F,T022A+S103A+S188D+N248D+E271F, T022A+V104I+S188D+A232V+Q245R,S103A+S188D+A232V+Q245R+N248D, S103A+S188D+Q245R+N248D+E271F,T022A+S103A+S188D+A232V+N248D, T022A+V104I+S188D+N248D+E271F,T022A+V104I+S188D+Q245R+E271F, S188D+A232V+Q245R+E271F,S188D+A232V+N248D+E271F, T022A+S103A+V104I+S188D+A232V+Q245R,V104I+S188D+A232V+Q245R+N248D, T022A+V104I+S188D+A232V+N248D,V104I+S188D+Q245R+N248D+E271F, T022A+S188D+A232V+E271F,T022A+S103A+V104I+S188D+Q245R+E271F,T022A+S103A+V104I+S188D+N248D+E271F, T022A+S188D+Q245R+N248D+E271F,S103A+S188D+A232V+Q245R+E271F, S103A+S188D+A232V+N248D+E271F,S103A+V104I+S188D+Q245R+N248D+E271F,T022A+S103A+V104I+S188D+A232V+N248D,S103A+V104I+S188D+A232V+Q245R+N248D, T022A+S103A+S188D+A232V+E271F,V104I+S188D+A232V+N248D+E271F, V104I+S188D+A232V+Q245R+E271F,T022A+S103A+S188D+Q245R+N248D+E271F, T022A+V104I+S188D+A232V+E271F,T022A+S188D+A232V+N248D+E271F, T022A+S188D+A232V+Q245R+N248D,S188D+A232V+Q245R+N248D+E271F, T022A+V104I+S188D+Q245R+N248D+E271F,S103A+V104I+S188D+A232V+N248D+E271F,S103A+V104I+S188D+A232V+Q245R+E271F, T022A+S188D+A232V+Q245R+E271F,T022A+S103A+V104I+S188D+A232V+E271F,T022A+S103A+S188D+A232V+N248D+E271F,T022A+S103A+S188D+A232V+Q245R+N248D,S103A+S188D+A232V+Q245R+N248D+E271F,T022A+V104I+S188D+A232V+Q245R+N248D,T022A+S103A+S188D+A232V+Q245R+E271F,T022A+V104I+S188D+A232V+N248D+E271F,T022A+S188D+A232V+Q245R+N248D+E271F,V104I+S188D+A232V+Q245R+N248D+E271F,T022A+V104I+S188D+A232V+Q245R+E271F, G159D+S188D+Q245R+N248D,T022A+G159D+S188D, T022A+S103A+G159D+S188D, T022A+V104I+G159D+S188D,T022A+S103A+V104I+G159D+S188D, S103A+G159D+S188D+Q245R,V104I+G159D+S188D+Q245R, S103A+V104I+G159D+S188D+Q245R,S103A+G159D+S188D+N248D, V104I+G159D+S188D+N248D,S103A+V104I+G159D+S188D+N248D, G159D+S188D+A232V,S103A+G159D+S188D+A232V, V104I+G159D+S188D+A232V,S103A+V104I+G159D+S188D+A232V, T022A+G159D+S188D+Q245R,G159D+S188D+E271F, T022A+S103A+G159D+S188D+Q245R,S103A+G159D+S188D+E271F, T022A+V104I+G159D+S188D+Q245R,V104I+G159D+S188D+E271F, T022A+S103A+V104I+G159D+S188D+Q245R,S103A+V104I+G159D+S188D+E271F, T022A+G159D+S188D+N248D,T022A+S103A+G159D+S188D+N248D, T022A+V104I+G159D+S188D+N248D,S103A+G159D+S188D+Q245R+N248D, T022A+S103A+V104I+G159D+S188D+N248D,V104I+G159D+S188D+Q245R+N248D, S103A+V104I+G159D+S188D+Q245R+N248D,T022A+G159D+S188D+A232V, T022A+S103A+0159D+S188D+A232V,T022A+V104I+G159D+S188D+A232V, G159D+S188D+A232V+Q245R,T022A+S103A+V104I+0159D+S188D+A232V, S103A+0159D+S188D+A232V+Q245R,G159D+S188D+Q245R+E271F, V104I+G159D+S188D+A232V+Q245R,G159D+S188D+A232V+N248D, T022A+G159D+S188D+E271F,S103A+0159D+S188D+Q245R+E271F, G159D+S188D+N248D+E271F,S103A+V104I+G159D+S188D+A232V+Q245R, S103A+0159D+S188D+A232V+N248D,T022A+G159D+S188D+Q245R+N248D, T022A+S103A+0159D+S188D+E271F,V104I+G159D+S188D+Q245R+E271F, T022A+V104I+G159D+S188D+E271F,V104I+G159D+S188D+A232V+N248D, S103A+0159D+S188D+N248D+E271F,T022A+S103A+0159D+S188D+Q245R+N248D,S103A+V104I+0159D+S188D+Q245R+E271F, V104I+G159D+S188D+N248D+E271F,G159D+S188D+A232V+E271F, T022A+S103A+V104I+G159D+S188D+E271F,S103A+V104I+G159D+S188D+A232V+N248D,T022A+V104I+G159D+S188D+Q245R+N248D,S103A+V104I+G159D+S188D+N248D+E271F, S103A+0159D+S188D+A232V+E271F,V104I+G159D+S188D+A232V+E271F, T022A+0159D+S188D+N248D+E271F,T022A+0159D+S188D+A232V+Q245R, T022A+G159D+S188D+Q245R+E271F,S103A+V104I+0159D+S188D+A232V+E271F, G159D+S188D+Q245R+N248D+E271F,T022A+S103A+0159D+S188D+A232V+Q245R,T022A+S103A+0159D+S188D+Q245R+E271F,T022A+S103A+0159D+S188D+N248D+E271F, T022A+G159D+S188D+A232V+N248D,G159D+S188D+A232V+Q245R+N248D, T022A+V104I+G159D+S188D+Q245R+E271F,T022A+V104I+G159D+S188D+N248D+E271F,T022A+V104I+G159D+S188D+A232V+Q245R,S103A+0159D+S188D+Q245R+N248D+E271F, G159D+S188D+A232V+Q245R+E271F,G159D+S188D+A232V+N248D+E271F, T022A+S103A+0159D+S188D+A232V+N248D,V104I+G159D+S188D+Q245R+N248D+E271F,S103A+0159D+S188D+A232V+Q245R+N248D, T022A+G159D+S188D+A232V+E271F,T022A+G159D+S188D+Q245R+N248D+E271F,S103A+0159D+S188D+A232V+Q245R+E271F,T022A+V104I+G159D+S188D+A232V+N248D,V104I+G159D+S188D+A232V+Q245R+N248D,S103A+G159D+S188D+A232V+N248D+E271F,T022A+S103A+0159D+S188D+A232V+E271F,V104I+G159D+S188D+A232V+Q245R+E271F,V104I+G159D+S188D+A232V+N248D+E271F,T022A+V104I+G159D+S188D+A232V+E271F,G159D+S188D+A232V+Q245R+N248D+E271F,T022A+G159D+S188D+A232V+N248D+E271F,T022A+G159D+S188D+A232V+Q245R+E271F,T022A+G159D+S188D+A232V+Q245R+N248D, S101G+S103A+S188D,S101G+V104I+S188D, S101G+S103A+V104I+S188D (List 9), and wherein aminoacid positions of the subtilisin variant are numbered according to thenumbering of corresponding amino acid positions in the amino acidsequence of Bacillus amyloliquefaciens subtilisin BPN′ shown in SEQ IDNO:1.

The present invention provides an isolated subtilisin variant, whereinsaid subtilisin variant is a mature form having proteolytic activity andcomprises an amino acid sequence comprising a combination of amino acidsubstitutions at positions selected fromT022A+S103A+V104I+S188D+Q245R+N248D,T022A+S103A+V104I+S188D+A232V+Q245R,T022A+S103A+V104I+S188D+Q245R+E271F,T022A+S103A+V104I+S188D+N248D+E271F,S103A+V104I+S188D+Q245R+N248D+E271F,T022A+S103A+V104I+S188D+A232V+N248D,S103A+V104I+S188D+A232V+Q245R+N248D,T022A+S103A+S188D+Q245R+N248D+E271F,T022A+V104I+S188D+Q245R+N248D+E271F,S103A+V104I+S188D+A232V+N248D+E271F,S103A+V104I+S188D+A232V+Q245R+E271F,T022A+S103A+V104I+S188D+A232V+E271F,T022A+S103A+S188D+A232V+N248D+E271F,T022A+S103A+S188D+A232V+Q245R+N248D,S103A+S188D+A232V+Q245R+N248D+E271F,T022A+V104I+S188D+A232V+Q245R+N248D,T022A+S103A+S188D+A232V+Q245R+E271F,T022A+V104I+S188D+A232V+N248D+E271F,T022A+S188D+A232V+Q245R+N248D+E271F,V104I+S188D+A232V+Q245R+N248D+E271F,T022A+V104I+S188D+A232V+Q245R+E271F,T022A+S103A+V104I+G159D+S188D+Q245R,T022A+S103A+V104I+G159D+S188D+N248D,S103A+V104I+G159D+S188D+Q245R+N248D,T022A+S103A+V104I+G159D+S188D+A232V,S103A+V104I+G159D+S188D+A232V+Q245R,T022A+S103A+G159D+S188D+Q245R+N248D,S103A+V104I+G159D+S188D+Q245R+E271F,T022A+S103A+V104I+0159D+S188D+E271F,S103A+V104I+0159D+S188D+A232V+N248D,T022A+V104I+G159D+S188D+Q245R+N248D,S103A+V104I+G159D+S188D+N248D+E271F,S103A+V104I+G159D+S188D+A232V+E271F,T022A+S103A+G159D+S188D+A232V+Q245R,T022A+S103A+G159D+S188D+Q245R+E271F,T022A+S103A+G159D+S188D+N248D+E271F,T022A+V104I+G159D+S188D+Q245R+E271F,T022A+V104I+0159D+S188D+N248D+E271F,T022A+V104I+0159D+S188D+A232V+Q245R,S103A+G159D+S188D+Q245R+N248D+E271F,T022A+S103A+G159D+S188D+A232V+N248D,V104I+G159D+S188D+Q245R+N248D+E271F,S103A+G159D+S188D+A232V+Q245R+N248D,T022A+G159D+S188D+Q245R+N248D+E271F,S103A+G159D+S188D+A232V+Q245R+E271F,T022A+V104I+G159D+S188D+A232V+N248D,V104I+G159D+S188D+A232V+Q245R+N248D,S103A+G159D+S188D+A232V+N248D+E271F,T022A+S103A+G159D+S188D+A232V+E271F,V104I+G159D+S188D+A232V+Q245R+E271F,V104I+0159D+S188D+A232V+N248D+E271F,T022A+V104I+G159D+S188D+A232V+E271F,G159D+S188D+A232V+Q245R+N248D+E271F,T022A+G159D+S188D+A232V+N248D+E271F,T022A+G159D+S188D+A232V+Q245R+E271F, orT022A+G159D+S188D+A232V+Q245R+N248D (List 10), and wherein amino acidpositions of the subtilisin variant are numbered according to thenumbering of corresponding amino acid positions in the amino acidsequence of Bacillus amyloliquefaciens subtilisin BPN′ shown in SEQ IDNO:1.

The present invention further provides an isolated subtilisin variant,wherein said subtilisin variant is a mature form having proteolyticactivity and comprises an amino acid sequence comprising a combinationof amino acid substitutions at positions selected fromS103A+V104I+S188D+A232V+Q245R, T022A+S103A+V104I+S188D+Q245R,T022A+S103A+V104I+S188D+N248D, S103A+V104I+S188D+Q245R+N248D,T022A+S103A+V104I+S188D+A232V, T022A+S103A+S188D+Q245R+N248D,T022A+V104I+S188D+Q245R+N248D, S103A+V104I+S188D+Q245R+E271F,S103A+V104I+S188D+A232V+N248D, T022A+S103A+V104I+S188D+E271F,S103A+V104I+S188D+N248D+E271F, S103A+V104I+S188D+A232V+E271F,T022A+S103A+S188D+A232V+Q245R, T022A+S103A+S188D+Q245R+E271F,T022A+S103A+S188D+N248D+E271F, T022A+V104I+S188D+A232V+Q245R,S103A+S188D+A232V+Q245R+N248D, S103A+S188D+Q245R+N248D+E271F,T022A+S103A+S188D+A232V+N248D, T022A+V104I+S188D+N248D+E271F,T022A+V104I+S188D+Q245R+E271F, V104I+S188D+A232V+Q245R+N248D,T022A+V104I+S188D+A232V+N248D, V104I+S188D+Q245R+N248D+E271F,T022A+S188D+Q245R+N248D+E271F, S103A+S188D+A232V+Q245R+E271F,S103A+S188D+A232V+N248D+E271F, T022A+S103A+S188D+A232V+E271F,V104I+S188D+A232V+N248D+E271F, V104I+S188D+A232V+Q245R+E271F,T022A+V104I+S188D+A232V+E271F, T022A+S188D+A232V+N248D+E271F,T022A+S188D+A232V+Q245R+N248D, S188D+A232V+Q245R+N248D+E271F,T022A+S188D+A232V+Q245R+E271F, T022A+S103A+V104I+G159D+S188D,S103A+V104I+G159D+S188D+Q245R, S103A+V104I+G159D+S188D+N248D,S103A+V104I+G159D+S188D+A232V, T022A+S103A+G159D+S188D+Q245R,T022A+V104I+G159D+S188D+Q245R, S103A+V104I+G159D+S188D+E271F,T022A+S103A+G159D+S188D+N248D, T022A+V104I+G159D+S188D+N248D,S103A+G159D+S188D+Q245R+N248D, V104I+G159D+S188D+Q245R+N248D,T022A+S103A+G159D+S188D+A232V, T022A+V104I+G159D+S188D+A232V,S103A+G159D+S188D+A232V+Q245R, V104I+G159D+S188D+A232V+Q245R,S103A+G159D+S188D+Q245R+E271F, S103A+G159D+S188D+A232V+N248D,T022A+G159D+S188D+Q245R+N248D, T022A+S103A+G159D+S188D+E271F,V104I+G159D+S188D+Q245R+E271F, T022A+V104I+G159D+S188D+E271F,V104I+G159D+S188D+A232V+N248D, S103A+G159D+S188D+N248D+E271F,V104I+G159D+S188D+N248D+E271F, S103A+G159D+S188D+A232V+E271F,V104I+G159D+S188D+A232V+E271F, T022A+G159D+S188D+N248D+E271F,T022A+G159D+S188D+A232V+Q245R, T022A+G159D+S188D+Q245R+E271F,G159D+S188D+Q245R+N248D+E271F, T022A+G159D+S188D+A232V+N248D,G159D+S188D+A232V+Q245R+N248D, G159D+S188D+A232V+Q245R+E271F,G159D+S188D+A232V+N248D+E271F, or T022A+G159D+S188D+A232V+E271F (List11), and wherein amino acid positions of the subtilisin variant arenumbered according to the numbering of corresponding amino acidpositions in the amino acid sequence of Bacillus amyloliquefacienssubtilisin BPN′ shown in SEQ ID NO:1.

The present invention further provides an isolated subtilisin variant,wherein said subtilisin variant is a mature form having proteolyticactivity and comprises an amino acid sequence comprising a combinationof amino acid substitutions at positions selected fromS103A+V104I+S188D+Q245R, T022A+S103A+V104I+S188D,S103A+V104I+S188D+A232V, S103A+V104I+0159D+S188D,S103A+V104I+S188D+N248D, S103A+S188D+A232V+Q245R,V104I+S188D+A232V+Q245R, T022A+S103A+S188D+Q245R,T022A+V104I+S188D+Q245R, S103A+V104I+S188D+E271F,T022A+S103A+S188D+N248D, T022A+V104I+S188D+N248D,S103A+S188D+Q245R+N248D, V104I+S188D+Q245R+N248D,T022A+S103A+S188D+A232V, T022A+V104I+S188D+A232V,T022A+S188D+Q245R+N248D, S103A+S188D+A232V+N248D,S103A+S188D+Q245R+E271F, V104I+S188D+A232V+N248D,V104I+S188D+Q245R+E271F, T022A+S103A+S188D+E271F,S103A+S188D+N248D+E271F, T022A+V104I+S188D+E271F,V104I+S188D+N248D+E271F, S103A+S188D+A232V+E271F,V104I+S188D+A232V+E271F, T022A+S188D+A232V+Q245R,T022A+S188D+Q245R+E271F, T022A+S188D+N248D+E271F,S188D+A232V+Q245R+N248D, S188D+Q245R+N248D+E271F,T022A+S188D+A232V+N248D, S188D+A232V+Q245R+E271F,S188D+A232V+N248D+E271F, T022A+S188D+A232V+E271F,G159D+S188D+Q245R+N248D, T022A+S103A+0159D+S188D,T022A+V104I+G159D+S188D, S103A+0159D+S188D+Q245R,V104I+G159D+S188D+Q245R, S103A+G159D+S188D+N248D,V104I+G159D+S188D+N248D, S103A+G159D+S188D+A232V,V104I+0159D+S188D+A232V, T022A+G159D+S188D+Q245R,S103A+0159D+S188D+E271F, V104I+G159D+S188D+E271F,T022A+0159D+S188D+N248D, T022A+G159D+S188D+A232V,G159D+S188D+A232V+Q245R, G159D+S188D+Q245R+E271F,G159D+S188D+A232V+N248D, T022A+G159D+S188D+E271F,G159D+S188D+N248D+E271F, G159D+S188D+A232V+E271F, orS101G+S103A+V104I+S188D (List 12), and wherein amino acid positions ofthe subtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention further provides an isolated subtilisin variant,wherein said subtilisin variant is a mature form having proteolyticactivity and comprises an amino acid sequence comprising a combinationof amino acid substitutions at positions selected fromS103A+V104I+S188D, S188D+Q245R+N248D, S103A+S188D+Q245R,V104I+S188D+Q245R, T022A+S103A+S188D, T022A+V104I+S188D,G159D+S188D+Q245R, S103A+S188D+A232V, S103A+G159D+S188D,V104I+S188D+A232V, V104I+0159D+S188D, S103A+S188D+N248D,V104I+S188D+N248D, G159D+S188D+N248D, S188D+A232V+Q245R,T022A+S188D+Q245R, S103A+S188D+E271F, V104I+S188D+E271F,T022A+S188D+N248D, T022A+S188D+A232V, S188D+A232V+N248D,S188D+Q245R+E271F, T022A+S188D+E271F, S188D+N248D+E271F,S188D+A232V+E271F, T022A+0159D+S188D, G159D+S188D+A232V,G159D+S188D+E271F, S101G+S103A+S188D, or S101G+V104I+S188D (List 13),and wherein amino acid positions of the subtilisin variant are numberedaccording to the numbering of corresponding amino acid positions in theamino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ shownin SEQ ID NO: 1.

The present invention further provides an isolated subtilisin variant,wherein said subtilisin variant is a mature form having proteolyticactivity and comprises an amino acid sequence comprising a combinationof amino acid substitutions at positions selected fromS101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S101G-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S101G-S103A-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S101G-S103A-V104I-S188D-A232V-Q245R-N248D-E271F,T022A-S101G-S103A-V104I-G159D-S188D-Q245R-N248D-E271F,T022A-S101G-S103A-V104I-G159D-S188D-A232V-N248D-E271F,T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-E271F,T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D,S101G-S103A-V104I-N116L-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S103A-V104I-N116L-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S101G-V104I-N116L-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S101G-S103A-N116L-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S101G-S103A-V104I-N116L-S188D-A232V-Q245R-N248D-E271F,T022A-S101G-S103A-V104I-N116L-G159D-A232V-Q245R-N248D-E271F,T022A-S101G-S103A-V104I-N116L-G159D-S188D-Q245R-N248D-E271F,T022A-S101G-S103A-V104I-N116L-G159D-S188D-A232V-N248D-E271F,T022A-S101G-S103A-V104I-N116L-G159D-S188D-A232V-Q245R-E271F,T022A-S101G-S103A-V104I-N116L-G159D-S188D-A232V-Q245R-N248D-E271F,G020R-N062E-S078G-G118S-S188D-N248D-H249R,S024R-N062E-G118R-A158E-S188D, T022A-S024R-T033S-G118R-S166D-S188D,S024R-N062E-S078G-G118S-S188D-Q245R-N248D,G020R-S024R-N062E-S078D-G118S-P129E-G159D,G020R-S024R-N062E-S078G-G118D-P129E,G020R-S024R-N062E-S078G-G118S-P129E-G159D-S188D-Q245R-N248D,G020R-N062E-S078G-G118S-G159D-Q245R-N248D, S024R-N116L-A158E-S166D,G020R-N062E-S078G-G118S-G159D-S188D-H249R,G020R-S024R-S078G-G118D-P129E-G159D-S188D, S024R-G118R-S166D,T022A-S024R-T033S-G118R-A158E-S166D-A273V,G020R-N062E-S078G-G118S-P129E-G159D-S188D-Q245R,T022A-G118R-A158E-S166D,G020R-S024R-N062E-S078D-G118S-G159D-S188D-Q245R-N248D,T022A-S024R-G118R-S166D-S188D, N062E-S078G-G118S-G159D,T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248E-E271H,G020R-S024R-N062E-S078G-G118D-Q245R-N248D,S024R-T033S-N116L-A158E-S166D,S024R-N062E-S078G-G118D-P129E-G159D-Q245R,G020R-N062E-S078D-G118S-Q245R, T022A-S024R-N062E-N116L-A158E,G020R-N062E-S078G-G118D-P129E, S024R-T033S-N062E-N116L-G118R-S188D,G020R-N062E-S078G-G118D-P129E-G159D-S188D-H249R,S024R-S078G-G118S-P129E-G159D-Q245R-N248D,G020R-N062E-S078G-G118D-S188D-H249R,T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245T-N248D-E271T,S078G-G118S-P129E-G159D-S188D-Q245R-N248D-H249R,T022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245T-N248H-E271T,G020R-S024R-S078D-G118S-G159D-S188D-H249R,G020R-S078D-G118S-P129E-G159D-Q245R-N248D-H249R,S024R-N062E-N116L-G118R, T022A-S024R-N116L-G118R-A158E-S188D,S024R-N062E-S078G-G118S-P129E-Q245R, S024R-N062E-G118R-A158E,G020R-N062E-S078G-G118S-G159D-S188D,G020R-S024R-S078D-G118S-G159D-S188D-Q245R-N248D-H249R,S024R-S078D-G118S-P129E-G159D-Q245R, A098Q-S099T-G102A-S103G,T022A-G118R-S166D-S188D, S024R-S078D-G118S-G159D-S188D-H249R,T022A-S024R-N116L-G118R-S166D-S188D, T022A-N062E-G118R-A158E,G020R-S078G-G118D-G159D-S188D-Q245R,S024R-N062E-S078G-G118D-G159D-S188D-Q245R,T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248E-E271F,T022A-S101G-S103A-V104I-G159D-S188E-A232S-Q245R-N248E-E271F,G020R-N062E-S078G-G118S-P129E-N248D-H249R,S024R-N116L-G118R-S128I-S166D-S188D,G020R-S024R-N062E-S078G-G118D-P129E-G159D-S188D-H249R,G020R-S024R-N062E-S078G-G118D-G159D-S188D-N248D,G020R-S024R-S078G-G118D-P129E-S188D-Q245R-N248D,S024R-N062E-S078D-G118S-S188D-H249R, T033S-N062E-G118R,T022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245T-N248E-E271H,T033S-G118R-A158E-S166D,T022Q-S101G-S103A-V104I-G159D-S188E-A232S-Q245V-N248H,G020R-S024R-S078D-G118D-S188D-Q245R-N248D-H249R, T022A-S024R-N062E,G020R-S024R-N062E-S078D-G118D-G159D-S188D-R247L,T022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245R-N248E-E271T,G020R-N062E-S078G-G118S-P129E-G159D-Q245R-N248D,T022Q-S101G-S103A-V104I-G159D-S188E-A232S-Q245R-N248H,P014S-G020R-S024R-S078G-G118S-P129E-S188D-Q245R-N248D,T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245R-N248E-E271L,G020R-S078D-G118D-G159D-S188D-H249R,T022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245R-N248D-E271T,T022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245T-N248E-E271L,S024R-N062E-S078D-G118S-S188D-Q245R,T022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245R-N248E-E271H,G020R-S078D-G118S-P129E-G159D-H249R,T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245R-N248H-E271F,T022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245R-N248H-E271H,S024F-T033S-S101G-S103A-V104I-N116A-Y209A-T213A-A232V,S024F-S101G-S103A-V104I-N116L-S128N-Y209V-G211Q-T213A-A232V,S024R-S078G-G118S-G159D-S188D-Q245R-N248D,G020R-S024R-S078G-G118S-S188D-Q245R,G020R-S024R-N062E-S078D-G118D-P129E-G159D-Q245R,S024R-S078D-G118D-G159D-S188D-H249R,G020R-S078G-G118S-G159D-S188D-H249R,G020R-S024R-N062E-S078G-G118S-S188D-N248D, 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G020R-S024R-S078D-G118S-G159D-Q245R-N248D,G020R-S024R-N062E-S078G-G118D-G159D-H249R,G020R-S024R-S078G-G118D-P129E-G159D-S188D-Q245R-N248D,T022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245T-N248D-E271H,N062E-S078G-G118S-N248D, T022A-S024R-N062E-S128I,G020R-S024R-N062E-S078D-G118D-G159D-S188D-Q245R-N248D,G020R-S024R-N062E-S078G-G118D-P129E-G159D-H249R,G020R-S024R-S078D-G118D-S188D-N248D-H249R,S024R-S078G-G118D-G159D-S188D,S024F-S101G-S103A-V104I-N116A-Y209A-G211Q-A232V,T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245R-N248E-E271L,T022A-S024R-T033S-N062E-G118R-S188D,G020R-S078D-G118D-P129E-G159D-S188D-Q245R-H249R,T022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245R-N248D-T255M-E271T,S024R-N062E-S078D-G118S-N248D-H249R,T022A-S101G-S103A-V104I-G159D-S188E-A232L-Q245R-N248D-E271H,G020R-S024R-S078D-G118D-P129E-G159D-S188D-H249R,T022Q-S101G-S103A-V104I-G159D-S188E-A232S-Q245T-N248D-E271L,G020R-S024R-N062E-S078G-G118D-P129E-Q245R-N248D,T022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245R-N248H-E271F,S024R-G118R-S128I-A158E-S188D,S024R-N062E-S078G-G118S-P129E-G159D-S188D-Q245R-N248D-H249R,T022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245V-N248H,T022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245T-N248E-E271H,T022A-G118R-S128I-A158E-S188D,T022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245R-N248E-E271T,S078G-G118S-P129E-G159D-S188D-Q245R-H249R,S024R-N062E-S078G-G118D-P129E-G159D, N116L-G118R-S128I-A158E,T022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245R-N248H-E271T,G020R-S024R-S078G-G118D-P129E-G159D-S188D-N248D-H249R,G020R-S024R-N062E-S078D-G118S-G159D-Q245R-N248D,S024R-S078D-G118S-P129E-G159D-Q245R-H249R,S024R-N062E-S078D-G118D-G159D, S024R-T033S-G118R-S188D,T022A-S101G-S103A-V104I-G159D-S188E-A232L-Q245R-N248H-E271L,T022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245V-N248E-E271L,S024R-S078G-G118D-P129E-S188D, S078G-G118D-G159D-S188D-H249R,T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245T-N248H-E271H,T022A-S101G-S103A-V104I-G159D-S188D-A232T-Q245V-N248D-E271L,A016S-T033S-S101G-S103A-V104I-N116A-Y209V-G211Q-T213A-A232V,T022A-S024R-N062E-N116L-S128I-A158E,T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245T-N248H-E271F,S024R-G118R-S128I-S188D, G020R-S078G-G118D-G159D-Q245R-N248D-H249R,N062E-S101A-S103A-V104I-S188D-P210I-A232V-Q245R,S024R-N062E-S078G-G118S-P129E-G159D-S188D-Q245R-N248D,S024R-N062E-S078G-G118D-N248D-H249R,S024R-N062E-S078G-G118S-G159D-S188D-N248D,S024R-N062E-S078G-G118S-P129E-S188D-Q245R, T022A-T033S-G118R-S166D,T022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245R-N248E-E271T,5078G-G118D-S188D-Q245R-N248D-H249R, T022A-S024R-G118R-S128I,T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245T-N248E-E271L,G020R-N062E-S078D-G118S-S188D,T022A-T033S-S101G-S103A-V104I-N116L-G159D-S188D-T213A-A232V-Q245R-N248D-E271F,T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245V-N248H-E271T,S024R-S078D-G118S-P129E-G159D-N248D-H249R,G020R-S078G-G118S-G159D-N248D-H249R, G020R-N062E-S078G-G118S-S188D,T022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245R-N248D-E271T,T022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245T-N248H,G020R-S024R-N062E-S078D-G118S-P129E-G159D-Q245R-N248D,G020R-N062E-S078D-G118S-G159D-Q245R, 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T022A-T033S-N116L-G118R-A158E,G020R-S024R-S078G-G118S-P129E-G159D-S188D-Q245R,A001G-T022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245T-N248H-E271L,S024R-S078D-G118S-G159D-Q245R,G020K-T022L-S078N-G118R-S166D-T213A-L217E,S078G-G118S-P129E-S188D-Q245R,T022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245T-N248E,T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245V-N248E-E271F,S024R-N062E-S078G-G118D-H249R, T022Q-S101G-S103A-V104I-G159D-S88D-A232T-Q245T-N248H-E271L,T022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245V-N248E-E271F,G020R-S024R-N062E-S078D-G118S-P129E-S188D,T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245T-N248H-E271L,S024R-S078D-G118D-P129E-S188D-H249R,T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245V-N248D-E271H,G020R-S078G-G118D-G159D,T022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245R-N248D-E271L,T022A-N062E-G118R-S128I-S188D, T022A-N116L-G118R-A158E-S166D-S188D,S024R-N062E-N116L, T022A-S024R-N116L-S128I-S166D-S188D,T022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245T-N248D-E271F,N116L-A158E-S166D, 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S103A-V104I-S128N-L148I,G020R-S024R-N062E-S078D-G118D-P129E-S188D-H249R,T022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245R-N248H-E271H,S024R-G118R-S188D,T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245V-N248E-E271F,T033S-N062E-G118R-A158E-S188D,T022A-S024F-S101G-S103A-V104I-N116A-S128N-Y209V-G211Q-T213A-A232V,S024R-N062E-S078G-G118S-P129E-G159D-S188D-H249R,S078G-G118S-P129E-G159D-H249R,T022A-S101G-S103A-V104I-G159D-S188D-A232T-Q245V-N248E-E271H,N062E-N076D-S101A-S103A-V104I-A232V-Q245R, G020K-S024F-S078N-L217E,S103A-S128N-L148I,S024F-S101G-S103A-V104I-N116L-Y209A-G211Q-T213A-A232V,G020R-S024R-S078D-G118S-P129E-G159D-S188D-Q245R-N248D-H249R,G020R-N062E-S078D-G118D-P129E-G159D-H249R,S078G-G118D-P129E-G159D-Q245R-H249R,G020R-N062E-S078D-G118S-G159D-Q245R-N248D-H249R,G020R-N062E-S078G-G118S-N248D-H249R,T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245R-N248E-E271L,G020R-S024R-S078D-G118D-G159D-S188D, T033S-N116L-A158E,G020K-S024F-S166D-L217E, S024R-S078D-G118D-G159D-S188D-Q245R-H249R,G020R-S024R-N062E-S078G-G118S-G159D-N248D-H249R,T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245T-N248H-E271H,T022A-S024R-T033S-G118R-A158E-S166D, N062E-S078D-G118D-G159D-Q245R,S078G-G118S-P129E-G159D-S188D-H249R,G020R-N062E-S078G-G118S-P129E-G159D,T022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245T-N248H-E271F,S078D-G118S-G159D-H249R, S078G-G118D-P129E-G159D-H249R,G020R-S024R-S078D-G118D-P129E-S188D-N248D-H249R,S024R-N062E-S078G-G118S-G159D-S188D-H249R,G020R-S078D-G118S-G159D-S188D-Q245R,G020R-N062E-S078D-G118S-G159D-S188D-H249R,G020R-S024R-N062E-S078G-G118S-P129E-Q245R-N248D,G020R-N062E-S078G-G118D-S188D-Q245R-N248D,S024R-N062E-S078D-G118S-G159D-S188D,G020R-S024R-N062E-S078G-G118S-P129E-Q245R,G020R-S024R-S078D-G118D-G159D,G020R-S078G-G118S-G159D-S188D-N248D-H249R,T022Q-S101G-S103A-V104I-G159D-S188E-A232T-Q245T-N248H,T022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245R-N248E-E271L,T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248H,T022A-S024R-N116L-G118R-S128I-S166D-S188D,S024R-N062E-S078G-G118S-G159D-S188D-Q245R-N248D,A016S-S024F-S101G-S103A-V104I-N116L-S128N-Y209V-G211Q-A232V,T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245V-N248H-E271L,S024R-S078G-G118S-P129E-S188D-Q245R-N248D-H249R,S024R-S078D-G118D-P129E-Q245R-H249R,T022Q-S101G-S103A-V104I-G159D-S188E-A232S-Q245V-N248D-E271F,G020K-S024F-S078N-G118R-S166D-L217E, S024F-G118R-S166D-T213A-L217E,G020R-S024R-S078D-G118S-P129E-S188D-Q245R-N248D-H249R:T022A-S024R-T033S-S078N-S166D-S188D, G020R-S024R-S078G-G118D-Q245R,G020K-S024F-S078N-G118R-S166D-T213A-L217E,G020K-T022L-S078G-G118S-G159D-S188D-H249R,T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245T-N248D-E271L,S024R-T033S-G118R-A158E-S166D-S188D,T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245T-N248D-E271L,T022V-S024F-T033S-S101G-S103A-V104I-N116L-Y209A-G211Q-T213A-A232V,G020R-S024R-S078G-G118D-G159D-S188D, S078N-G118R-S166D-L217E,S024R-S078G-G118S-G159D-Q245R-N248D,T022A-S024R-G118R-S128I-A158E-S166D-S188D,T022A-S101G-S103A-V104I-G159D-S188E-A232L-Q245R-N248H-E271H,T022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245T-N248H-E271T,G020R-S024R-S078G-G118D-G159D-N248D-H249R,T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245V-N248D-E271F,A016S-T022A-S101G-S103A-V104L-L148I,G020R-S024R-N062E-S078D-G118S-G159D-N248D,T022Q-S101G-S103A-V104I-G159D-S188E-A232T-Q245R-N248E-E271H,S078G-G118D-P129E-G159D-Q245R, S024R-S078G-G118S-S188D-Q245R-N248D,G020R-N062E-S078G-G118D-G159D-S188D,G020R-S024R-N062E-S078D-G118D-G159D-S188D-Q245R,T022A-T033S-S101G-S103A-V104I-N116L-Y209A-G211Q-T213A-A232V,T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248H-E271H,T022A-S024R-T033S-S128I-S188D,T022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245T-N248D-E271H,T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245R-N248E-E271F,S024R-S078G-G118S-P129E-S188D-N248D,G020R-N062E-S078D-G118S-P129E-Q245R,T022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245T-N248D-E271F,T022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245T-N248H,G020R-S024R-S078D-G118D-G159D-Q245R,T022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245T-N248E-E271L,S101A-N116L-P210I-T213A,T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248H-E271F,G020R-S024R-N062E-S078D-G118D-P129E-Q245R-N248D,T022A-S024R-N062E-A158E,T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245T-N248D-E271H,G020R-S024R-N062E-S078D-G118D-G159D-S188D-Q245R-N248D-H249R,G020R-N062E-S078G-G118S-G159D-S188D-Q245R-N248D,N062E-S078G-G118S-S188D-H249R,A016S-S024F-T033S-S101G-S103A-V104I-N116L-Y209V-G211Q-T213A-A232V,G020R-S078G-G118D-S188D-H249R,T022A-S024F-S101G-S103A-V104I-N116L-S128N-Y209A-G211Q-A232V,G020R-N062E-S078D-G118S-G159D-N248D-H249R,T022A-S101G-S103A-V104I-G159D-S188D-A232T-Q245T-N248D-E271H,T022A-S024R-N116L-G118R-A158E-S166D-S188D,G020R-S024R-S078G-G118S-P129E-G159D-S188D-Q245R-N248D-H249R,S024R-N062E-N076D-S101A-S103A-V104I-P210I-A232V-Q245R,N062E-S078D-G118S-G159D-Q245R-N248D-H249R,G020R-S024R-S078G-G118D-G159D-S188D-N248D-H249R,T022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245V-N248E-E271T,T022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245V-N248E-E271T,S024R-S101A-S103A-V104I-S188D-P210I-A232V-Q245R,S024R-N062E-N116L-G118R-A158E, G020R-S078G-G118D-S188D-Q245R,T022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245V-N248E-E271L,T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245R-N248E-E271H,S024R-S166D-S188D,T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245T-N248D-E271F,A016S-T022A-S024F-S101G-S103A-V104I-N116A-Y209V-G211Q-A232V,T022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245T-N248D-E271F,G020R-N062E-S078G-G118D-G159D-S188D-Q245R-N248D,T022A-N062E-N116L-G118R-A158E, S024R-N062E-N116L-G118R-S128I-S166D,T022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245R-N248H,G020R-S024R-N062E-S078D-G118D-H249R,T022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245R-N248E-E271L,S024R-N062E-G118R-S128I-S188D, N062E-S078G-G118S-G159D-N248D-H249R,G020K-T022L-N062E-S078D-G118S-G159D-H249R,N062E-S078G-G118D-Q245R-N248D,G020R-S024R-N062E-S078D-G118D-G159D-S188D-H249R,T022A-S024F-S101G-S103A-V104I-N116L-Y209V-T213A-A232V,T022A-S101G-S103A-V104I-G159D-S188D-A232T-Q245R-N248D-E271L,S024R-N116L-G118R-S128I-A158E,T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245R-N248E-E271F,T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245V-N248E-E271T,T022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245V-N248D-E271L,A016S-S101G-S103A-V104L-L148I,T022Q-S101G-S101G-S103A-V104I-G159D-S188E-A232L-Q245T-N248H-E271T,G020R-N062E-S078G-G118S-S188D-Q245R,T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245V-N248E-E271H,S024R-S078D-G118D-S188D-Q245R-N248D-H249R,G020R-S024R-S078D-G118S-P129E-Q245R-N248D,G020R-S078G-G118D-G159D-S188D,T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245T-N248E-E271T,G020R-S024R-S078D-G118D-G159D-N248D-H249R,S024R-N062E-S078G-G118S-P129E-S188D-Q245R-N248D,T022A-S101G-S103A-V104I-G159D-S188E-A232L-Q245T-N248D-E271T,T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245R-N248E,T022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245T-N248H-E271F,T022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245T-N248E-E271F,T022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245R-N248D-E271L,G020R-S024R-N062E-S078D-G118S-P129E-N248D,S024R-N062E-S078D-G118S-P129E-G159D-N248D-H249R,T022Q-S101G-S103A-V104I-G159D-S188E-S212L-A232S-Q245V-N248D-E271T,S024R-S078G-G118D-G159D-H249R, T022A-N116L-G118R-S128I-S188D,T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245R-N248D,N062E-S078G-G118D-S188D-Q245R, G020K-S078N-S166D-L217E,N062E-S078G-G118S-P129E-S188D-Q245R,T022Q-S101G-S103A-V104I-G159D-S188E-A232S-Q245V-N248E-E271F,N062E-N116L-G118R-S166D-S188D, G020R-S024R-S078D-G118S-G159D-N248D,T022A-S024F-S101G-S103A-V104I-N116L-S128N-Y209V-A232V,S078G-G118S-P129E-G159D-Q245R,T022A-T033S-S101G-S103A-V104I-N116L-Y209V-G211Q-T213A-A232V,S078D-G118S-P129E-G159D-S188D-Q245R-N248D-H249R,S101G-S103A-V104I-N116L-S128N-Y209A-G211Q-T213A-A232V,G097P-S101A-S103G, S024R-N062E-S078D-G118S-S188D,G020R-S024R-S078D-G118D-P129E-S188D-H249R,S024R-N062E-S078G-G118S-G159D,A016S-T022A-T033S-S101G-S103A-V104I-N116A-Y209A-A232V,T022A-N116L-G118R-S128I-A158E-S166D,T022Q-S101G-S103A-V104I-G159D-S188E-A232T-Q245R-N248D-E271T,S024R-G118R-S128I,T022A-S101G-S103A-V104I-G159D-S188E-A232L-Q245T-N248E-E271L,S024R-N062E-A158E, S024R-S078D-G118S-P129E,G020R-S024R-S078D-G118S-P129E-G159D-S188D-H249R,T022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245V-N248H-E271F,T022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245R-N248H-E271F,G020R-S024R-N062E-S078G-G118S-G159D,T022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245V-N248H-E271L,S024R-S078D-G118S-P129E-S188D-Q245R-N248D,N062E-S078G-G118S-S188D-Q245R-N248D, N062E-N116L-G118R-S128I-S188D,T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245V-N248D-E271F,G020R-S024R-S078D-G118S-G159D-S188D-Q245R-N248D,T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245T-N248D-E271T,T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245V-N248E-E271T,T022A-T033S-N116L-G118R-S188D,G020R-S024R-N062E-S078G-G118S-P129E-S188D-N248D,G020R-N062E-S078G-G118S-P129E-G159D-N248D,S078G-G118S-P129E-G159D-S188D-Q245R, N062E-S078D-G118S-G159D-H249R,T022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245R-N248D,T022A-S024R-T033S-N062E-N116L,T022Q-S101G-S103A-V104I-G159D-S188E-A232S-Q245T-N248E-E271T,N062E-S078D-G118S-H249R, T022A-S024R-N062E-N116L-G118R-A158E,T022L-S078N-G118R-S166D-L217E,G020R-S024R-N062E-S078D-G118D-S188D-Q245R-N248D-H249R,T022A-S101G-S103A-V104I-G159D-S188E-A232S-Q245R-N248H-E271L,G020R-S024R-S078G-G118D-P129E-Q245R-N248D-H249R,T022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245V-N248D-E271L,S078D-G118S-P129E-S188D-Q245R,S024R-S078G-G118D-G159D-S188D-Q245R-H249R,S078D-G118D-G159D-S188D-Q245R-H249R, S024F-T213A-L217E,S024F-T033S-S101G-S103A-V104I-N116L-S128N-Y209V-G211Q-A232V,T022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245T-N248D-E271H,T022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245T-N248E-E271T,T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248H-E271H,G020R-S078D-G118D-P129E-G159D-Q245R-H249R,T022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245R-N248D,T022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245R-N248D-E271T,N062E-S078G-G118S-P129E-Q245R, S078G-G118S-S188D-Q245R-N248D-H249R,S101A-S103G-V104I-L148I,T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245R-N248H-E271F,T022Q-S101G-S103A-V104I-G159D-S188E-A232T-Q245V-N248H-E271H,T022A-N116L-G118R-A158E,T022A-S024F-T033S-S101G-S103A-V104I-I107V-N116L-S128N-Y209A-G211Q-T213A-A232V,T022Q-S101G-S103A-V104I-G159D-S188E-A232S-Q245R-N248H-E271T,G020R-N062E-S078D-G118D-G159D, G020R-N062E-S078D-G118S,S024R-N062E-S078D-G118D-P129E-G159D-Q245R,T022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245V-N248E-E271T,T022A-S101G-S103A-V104I-G159D-S188D-A232T-Q245V-N248D-E271H,S078G-G118S-G159D, S024F-G118R-T213A-L217E,S024R-N062E-S078D-G118S-P129E-Q245R-N248D,G020R-S024R-S078D-G118S-P129E-G159D-Q245R,S024R-S078D-G118D-G159D-H249R,S024R-N076D-S101A-S103A-V104I-S188D-P210I-A232V-Q245R,G020R-S024R-N062E-S078D-G118D-P129E-G159D-H249R,G020R-S024R-N062E-S078G-G118D-S188D-N248D-H249R,G020R-S024R-N062E-S078G-G118S-P129E-S188D-H249R,T022Q-S101G-S103A-V104I-G159D-S188E-A232S-Q245V-N248D,T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245T-N248E-E271H,T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245V-N248D-E271T,G020R-S024R-S078D-G118D-P129E-G159D-N248D-H249R,G020R-S024R-N062E-S078D-G118S-P129E-G159D-N248D,N062E-S078G-G118D-P129E-H249R, G020K-T022L-S078N-G118R-T213A-L217E,G020R-S024R-N062E-S078G-G118D-P129E-S188D-H249R,T022A-S024R-T033S-N116L-S166D-S188D,S024R-S078G-G118D-G159D-S188D-H249R, T033S-N116L-G118R-A158E-S188D,T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245V-N248H,S101A-S103A-V104I-S188D-P210I-A232V-Q245R,G020R-S024R-N062E-S078G-G118D-S188D-Q245R,T022A-S024R-T033S-N062E-N116L-G118R-P129E-S188D,T022A-S101G-S103A-V104I-G159D-S188E-A232S-Q245T-N248E,S024R-S078G-G118S, N062E-S101A-S103A-V104I-P210I-A232V-Q245R,N062E-S078G-G118D-G159D,T022A-S024R-N062E-N116L-G118R-S128I-S166D-S188D,S024R-S078D-G118D-G159D-Q245R,G020R-N062E-S078G-G118S-G159D-S188D-N248D-H249R,T022Q-S101G-S103A-V104I-G159D-S188E-A232S-Q245R-N248E-E271H,T022Q-S101G-S103A-V104I-G159D-S188D-A232T-V244I-Q245R-N248H-E271L,S024R-S078G-G118S-P129E-G159D-N248D,S024R-N062E-S078G-G118S-G159D-H249R,T022A-S024F-T033S-S101G-S103A-V104I-N116L-Y209V-G211Q-A232V,A016S-S101G-S103A-V104I-N116A-S128N-Y209V-G211Q-T213A-A232V,G020R-N062E-S078G-G118D-S188D,T022A-S101G-S103A-V104I-G159D-S188E-A232S-Q245V-N248E-E271H,T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245V-N248E-E271L,G020K-S166D-L217E,T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245T-N248E-E271T,G020R-N062E-S078D-G118D-Q245R,S024R-N062E-S078D-G118S-P129E-S188D-Q245R-N248D,A016S-T022A-S024F-S101G-S103A-V104I-N116A-S128N-Y209A-G211Q-A232V,S024F-T033S-S101G-S103A-V104I-N116L-Y209A-A232V, T033S-G118R-S188D,T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245T-N248E-E271H,T022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245T-N248D-E271T,G020R-S024R-S078G-G118S-P129E-G159D-S188D-N248D,T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245T-N248H-E271F,G020R-S078G-G118D-P129E-S188D-Q245R,G020R-S078G-G118D-G159D-S188D-Q245R-N248D,S024R-S078G-G118D-P129E-N248D-H249R, G020R-S024R-S078D-G118D-P129E,N062E-S078D-G118S-S188D-Q245R, G020R-S024R-S078G-G118S-G159D-S188D,T022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245V-N248H-E271L,T022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245R-N248D-E271L,N062E-S078G-G118S-G159D-H249R, G020R-S078D-G118S-P129E-G159D-N248D,G020R-S024R-S078D-G118S-G159D-S188D-Q245R,G020R-N062E-S078G-G118D-P129E-S188D-Q245R-N248D,T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245R-N248H,G020R-S024R-S078D-G118S-G159D-V244L-Q245R-N248D,G020R-S024R-S078G-G118D-P129E-S188D-Q245R-N248D-H249R,T022A-S024F-T033S-S101G-S103A-V104I-N116A-Y209A-A232V,G020R-S024R-N062E-S078D-G118S-P129E-G159D-A272D,N116L-G118R-S128I-A158E-S166D, S024R-S078D-G118S-P129E-H249R,S024R-N062E-S078G-G118S-P129E-N248D,G020R-S078G-G118S-P129E-G159D-S188D,G020R-S024R-S078D-G118S-G159D-N248D-H249R,S024R-S078D-G118D-G159D-S188D-Q245R-N248D-H249R,T033S-S101G-S103A-V104I-N116L-Y209A-G211Q-T213A-A232V,T033S-S101G-S103A-V104I-N116A-Y209A-T213A-A232V,T022A-S101G-S103A-V104I-G159D-S188E-A232S-Q245V-N248H,N062E-S078G-G118S-N248D-H249R,G020R-N062E-S078D-G118D-G159D-S188D-Q245R,T022A-S024F-S101G-S103A-V104I-N116A-Y209A-T213A-A232V,N043F-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,S024R-S078G-G118D-P129E-G159D,G020R-N062E-S078D-G118D-G159D-Q245R-N248D, S024R-S166D,S024R-S078G-G118S-P129E-N248D,N076D-S101A-S103A-V104I-S188D-P210I-A232V-Q245R, G020K-L217E,G118R-S166D-S188D, S024R-S078D-G118S-G159D-S188D,S024R-S078D-G118S-P129E-S188D-Q245R,A016S-S101G-S103A-V104I-N116A-S128N-Y209A-T213A-A232V, N116L-S166D,S024R-N062E-S078G-G118D-P129E-G159D-S188D-Q245R,G020R-S024R-S078D-G118D-P129E-N248D-H249R,N062E-S078G-G118S-G159D-S188D-Q245R,T022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245V-N248E-E271H,S024R-N062E-S078D-G118S-P129E-H249R, G118R-S166D-T213A,G020R-S024R-N062E-S078G-G118S-P129E-G159D,G020R-S024R-S078D-G115W-G118D-G159D-N248D,A016S-S024F-S101G-S103A-V104I-N116A-Y209A-A232V,S024R-N062E-N116L-S128I-A158E,T022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245V-N248D-E271L,S024R-N062E-S078G-G118S-P129E-H249R,S024R-N062E-S078D-G118D-P129E-N248D-H249R,G020K-T022L-S024F-S078N-G118R-L217E, S101G-G102A-S103A,G020R-S078G-G118S-P129E-Q245R-N248D, G020K-S078N-S166D,T022A-G118R-A158E, G020K-T022L-S024F-S078N-S166D,T022A-T033S-N062E-N116L-G118R-A158E-S188D,S024R-S078G-G118S-P129E-G159D-H249R,S101G-S103A-V104I-N116L-Y209V-G211Q-A232V,A015D-A016S-S101G-S103A-V104I-N116L-S128N-Y209V-T213A-A232V,G020R-N062E-S078D-G118D-P129E-S188D-H249R, G020K-S024F-S078N-S128D,T033S-N116L-S166D-S188D, G020R-S078G-G118S-P129E-G159D,T022A-S024R-T033S-L082I-G118R-S128I-A158E-S188D,G020R-S024R-S078D-G118S-S188D-N248D,G020R-S078G-G118D-P129E-S188D-Q245R-H249R, N062E-S078G-G118D-Q245R,G020K-T022L-S078N-L217E,T022A-S101G-S103A-V104I-G159D-S188D-A232T-Q245V-N248E-E271L,T022Q-S101G-S103A-V104I-G159D-S188E-A232S-Q245V-N248E-E271H,T022Q-S101G-S103A-V104I-G159D-S188E-A232T-Q245T-N248E-E271T,A016S-T033S-S101G-S103A-V104I-N116L-Y209V-G211Q-T213A-A232V,T022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245K-N248H,N062E-S078D-G118D-Q245R, T022A-S024R-G118R-A158E-S166D,A016S-S101A-S103A-S128N-L148I,T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245R-N248E,S024R-N062E-S078G-G118D, S024R-N062E-S078G-G118S-P129E-G159D-S188D,S078G-G118D-P129E-G159D-S188D-Q245R-N248D-H249R,G020R-S078D-G118D-P129E-G159D-Q245R,T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245V-N248H,T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271H,G020R-S024R-N062E-S078G-G118D-H249R, S078D-G118S-P129E-G159D-H249R,T022Q-S101G-S103A-V104I-G159D-S188E-A232S-Q245T-N248D-E271H,S024R-S078D-G118D-P129E-G159D-S188D-Q245R, T022L-T213A-L217E,G020R-N062E-S078G-G118S-P129E-G159D-S188D-N248D-H249R,T022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245R-N248H-E271T,G020R-N062E-S078G-G118D-Q245R,G020R-S024R-N062E-S078G-G118D-N248D-H249R,T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248H-E271T,S024R-S078D-G118D-P129E-H249R, S024R-N062E-N116L-G118R-S188D,S024R-N062E-G118R,T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248E,G020R-S024R-S078G-G118S-P129E-G159D-S188D-H249R,T022A-S101G-S103A-V104I-G159D-S188E-A232L-Q245R-N248H,S024R-S078D-G118S-P129E-G159D-N248D,T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245V-N248D-E271T,G020R-S078G-G118D-P129E-Q245R-N248D-H249R,G020R-S024R-N062E-S078D-G118D-P129E-S188D-Q245R-N248D,A016S-S024F-T033S-S101G-S103A-V104I-N116L-Y209V-A232V,G097P-A098Q-S099G-V104L,T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245T-N248D-E271F,G020R-S024R-N062E-S078G-G118S-N248D, A016S-S103A-S128N,S024R-S078G-G118D-P129E-Q245R, S078D-G118D-P129E-S188D-H249R,T022A-S024R-T033S-N062E-N116L-A158E,A016S-T022A-S101G-S103A-V104I-N116A-S128N-Y209V-T213A-A232V,S024R-S078G-G118S-G159D-S188D-Q245R,T033S-N062E-N116L-G118R-A158E-S188D,G020R-S024R-N062E-S078D-G118S-G159D-Q245R-A272V,G118R-S128I-A158E-S166D, G020K-T022L-S024F-S078N-S166D-D181E-L217E,N062E-G118R-S188D,S024F-S101G-S103A-V104I-N116L-S128N-Y209A-T213A-A232V,G020R-S078D-G118D-G159D-S188D-Q245R,S024R-N062E-S078G-G118D-P129E-N248D-H249R,G020R-S024R-N062E-S078G-G118D-N248D,T022A-V068A-S101G-S103A-V104I-G159D-S188D-T213A-A232V-Q245R-N248D-E271F,G020R-N062E-S078D-G118S-P129E-G159D-S188D-Q245R,S024R-S078G-G118S-P129E-N248D-H249R,T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248H-E271L,T033S-N116L-G118R-S188D, N062E-S078G-G118S-S188D-N248D-H249R,A016S-T022A-S101G-S103A-V104I-S128N-L148I,G020R-S078G-G118S-S188D-N248D-H249R,G020R-S024R-S078D-G118D-P129E-G159D-Q245R-N248D,S024F-T033S-S101G-S103A-V104I-N116L-Y209A-T213A-A232V,S024R-N062E-G118R-S128I-A158E, G020R-S024R-S078D-G118S-G159D-S188D,S024R-N062E-S078G-G118D-G159D-Q245R-N248D-H249R,N062E-G118R-A158E-S166D, G020R-S024R-S078D-G118S-P129E-S188D,S078G-G118D-G159D,G020R-S024R-S078D-G118D-P129E-G159D-S188D-Q245R-N248D,G020R-S078G-G118D-S188D-N248D,G020K-T022L-S078G-G118D-G159D-N248D-H249R, S103G-L148I-M175T,G020R-S024R-S078G-G118S-P129E-Q245R-N248D,T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245R-N248D-H249R-E271F,T022A-S024F-S101G-S103A-V104I-N116A-S128N-Y209A-G211Q-T213A-A232V,A016S-T033S-S101G-S103A-V104I-N116A-Y209A-G211Q-A232V,T022A-S024R-N062E-S166D,G020R-S078D-G118S-G159D-S188D-Q245R-N248D-H249R,T022A-S024F-S101G-S103A-V104I-N116A-S128N-Y209V-A232V,T022A-S024R-N062E-N116L-A158E-S188D,T022Q-S101G-S103A-V104I-G159D-S188E-A232S-Q245T-N248E-E271L,S024R-T033S-N116L-G118R-A158E-S188D,T022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245V-N248H-E271H,G020R-N062E-S078D-G118S-G159D-N248D, T022A-S024R-N116L-G118R-A158E,G020K-T022L-S024F-G118R-L217E,S024R-S078D-G118S-G159D-S188D-N248D-H249R,G020R-S024R-N062E-S078G-G118D-P129E-S188D-N248D,T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271L,G020R-S024R-N062E-S078D-G118S-P129E-G159D-S188D-N248D-H249R,N062E-G118R-S166D,T022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245R-N248D,G020R-N062E-S078D-G118S-P129E-G159D-N248D-H249R,T022A-S101G-S103A-V104I-G159D-S188D-A232T-Q245V-N248H-E271T,T022L-S024F-G118R-S166D,T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D,G020K-S024F-S166D-T213A-L217E, N062E-S078G-G118S-G159D-S188D,S024R-N062E-S078D-G118D-Q245R-N248D,G020K-T022L-S024F-S078N-T213A-L217E,T022A-V068A-S101G-S103A-V104I-N116L-G159D-S188D-T213A-A232V-Q245R-N248D-E271F,N062E-S078G-G118S-P129E-G159D,G020R-S024R-N062E-S078G-G118S-G159D-H249R,G020K-T022L-S024F-S078N-G118R-S166D-T213A-L217E,T022A-N062E-N116L-A158E,T022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245V-N248E-E271L,T022A-S024R-N062E-S128I-S166D,S024R-S078G-G118D-G159D-Q245R-N248D-H249R,G020R-S024R-N062E-S078G-G118S-P129E-G159D-H249R,G020R-S078G-G118D-S188D-Q245R-N248D,G020R-S024R-N062E-S078G-G118D-P129E-S188D-Q245R-N248D-H249R,S024R-S078G-G118S-G159D-Q245R, T022A-S024R-N062E-G118R-S128I-A158E,G020R-S078D-G118S-P129E-G159D-S188D-Q245R,S024R-S078G-G118S-Q245R-N248D-H249R, G118R-A158E-S188D,G020K-S024F-G118R-S166D-L217E, G020K-S024F-S078N-G118R-T213A-L217E,T022A-S101G-S103A-V104I-G159D-S188E-A232S-Q245V-N248D-E271L,T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245V-N248D,S024R-N062E-S078G-G118S-H249R,G020R-S024R-S078G-G118S-G159D-S188D-Q245R-N248D-H249R,A016S-S024F-S101G-S103A-V104I-N116L-S128N-Y209V-A232V,T033S-A158E-S166D, S024R-S078D-G118S-S188D-N248D-H249R,S024R-N062E-S078D-G118S-S188D-Q245R-N248D,N062E-S078G-G118D-G159D-Q245R-N248D,N062E-S078G-G118D-S188D-Q245R-N248D-H249R,S024R-N116L-G118R-S128I-A158E-S166D,G020R-N062E-S078D-G118S-P129E-G159D-Q245R-N248D,S024R-N062E-S101A-S103A-V104I-S188D-P210I-A232V-Q245R,N062E-S078D-G118S-P129E-H249R, S024R-S078G-G118S-P129E-G159D-S188D,A016S-S101G-S103A-V104I-N116A-S128N-Y209V-T213A-A232V,G020K-T022L-S024F-S078N-S166D-T213A-L217E,G020R-S024R-N062E-S078D-G118D-S188D-N248D,G020R-S024R-N062E-S078G-G118S-G159D-A272D, T022A-S024R-A158E,G020R-S078G-G118S-G159D-N248D,T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245V-N248E-E271T,S101G-S103A-V104I-N116A-Y209A-G211Q-T213A-A232V,G020K-T022L-S024F-S166D-L217E, G020R-S024R-S078D-G118S-Q245R,G020R-S078D-G118D-P129E-S188D-Q245R,G020R-S024R-N062E-S078D-G118S-S188D-Q245R-N248D,G020R-S024R-S078D-G118D-G159D-N248D,T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245T-N248H,G020R-S024R-S078D-G118S-S188D-Q245R,0020R-N062E-S078D-G118D-S188D-Q245R,G020R-S024R-S078D-G118D-G159D-S188D-Q245R-H249R,T022A-S128I-A158E-S188D,A016S-S024F-S101G-S103A-V104I-N116A-S128N-Y209V-T213A-A232V-V244G,T022L-S078N-T213A-L217E, S078D-G118S-P129E-H249R,G020K-T022L-S024F-S078N-G118R-T213A-L217E,N062E-S078D-G118S-G159D-Q245R, G020K-S024F-S166D,S024R-N062E-S078G-G118D-Q245R-N248D-H249R,G020K-S024F-S078N-G118R-L217E, T022L-S024F-S078N-G118R-S166D-L217E,T022A-S024R-T033S-A158E-S188D,T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245T-N248D-E271H, S087R,T022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245V-N248H,G020R-S078G-G118D-P129E-G159D-Q245R-N248D-H249R,S078G-G118S-P129E-G159D-Q245R-N248D,S024R-S078D-G118D-P129E-G159D-Q245R-N248D-H249R,0020R-S078G-G118S-P129E, S024R-N116L-G118R-S166D-S188D,G020R-S024R-S078G-G118S-P129E-G159D-Q245R, T022A-S101G-S103G-V104I,G020R-S024R-N062E-S078D-G118D-P129E-G159D,G020R-S024R-N062E-S078D-G118D-P129E-Q245R-N248D-H249R,T022A-S024R-N062E-N116L-G118R-S188D,T022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245T-N248E-E271L,A016S-S101G-S103A-V104I-N116L-M119V-Y209V-G211Q-A232V,G020R-S024R-N062E-S078G-G118D,T022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245T-N248H-E271L,T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245T-N248D-E271T,T033S-S101G-S103A-V104I-N116L-Y209A-G211Q-A232V,G020R-S024R-N062E-S078D-G118S-P129E-G159D-N248D-H249R,G020R-S078G-G118S-P129E-N248D-H249R, G020K-T022L-G118R-S128D,T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245V-N248D-E271L,G020R-S024R-S078D-G118S-G159D-S188D-N248D-H249R,S101G-S103A-V104I-N116L-Y209A-T213A-A232V,T022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245T-N248E-E271T,S024R-N062E-S078D-G118D-G159D-Q245R-N248D-H249R,G020K-S024F-T213A-L217E, G118R-S128I-S188D,G020R-S078G-G118S-P129E-H249R, T022A-N062E-G118R-S188D,S024R-S078G-G118S-P129E-G159D-Q245R,T022A-S024F-S101G-S103A-V104I-N116L-Y209A-G211Q-T213A-A232V,T022A-S024R-N116L-A158E-S166D,T022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245T-N248H-E271T,T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245T-N248H-E271L,S024R-N062E-A158E-S188D, G020K-S024F-G118R-S166D-T213A-L217E,T022A-S101G-S103A-V104I-G159D-S188E-A232S-Q245T-N248H-E271T,S078D-G118D-P129E-G159D-Q245R, S101A-S103A-V104I-S188D-A232V-Q245R,N062E-N116L-G118R-A158E,T022A-T033S-S101G-S103A-V104I-N116L-Y209A-T213A-A232V,T022L-S024F-G118R-S128D-T213A,A016S-S024F-S101G-S103A-V104I-N116L-S128N-Y209A-A232V,S078D-G118S-G159D-S188D-N248D-H249R, G020R-S078G-G118S-P129E-S188D,T022A-T033S-N116L-S166D, S024R-N062E-G118R-S188D,S024R-T033S-N116L-G118R-S128I-A158E,S101A-S103A-V104I-P210I-A232V-Q245R,G020R-S024R-N062E-S078G-G118S-S188D-H249R,T022A-S024R-N116L-G118R-S128I-A158E-S166D-S188D,A016S-S024F-S101G-S103A-V104I-N116L-Y209V-G211Q-T213A-A232V,T022A-S101G-S103A-V104I-G159D-S188E-A232S-Q245V-N248D-E271H,S078G-G118S-G159D-S188D-N248D-H249R,S024R-S078D-G118D-P129E-G159D-S188D-H249R,T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245T-N248E-E271F,G020R-N062E-S078D-G118D, S024R-T033S-N116L-G118R-S128I-A158E-S188D,T033S-N062E-N116L-G118R-S188D, T022A-T033S-N116L-G118R-A158E-S188D,S078N-G118R-S166D,T022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245T-N248E-E271L,T022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245T-N248H-E271L,S024F-S101G-S103A-V104I-N116A-Y209A-G211Q-T213A-A232V,G020R-S024R-S078D-G118D-G159D-S188D-Q245R-N248D,T022A-S101G-S103A-V104I-N116L-G159D-S188D-T213A-A232V-Q245R-N248D-E271F,T022A-G118R-A158E-S188D, S024R-S078D-G118S-G159D-H249R,S101G-S103A-V104I-N116L-Y209A-G211Q-A232V,T022A-S101G-S103A-V104I-G159D-S188E-A232S-Q245V-N248E-E271F,S024R-N062E-S078D-G118S-P129E-Q245R, 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S024R-S078D-G118D-G159D-Q245R-N248D,A016S-T022A-S024F-S101G-S103A-V104I-N116L-S128N-Y209A-G211Q-T213A-A232V,S078G-G118D-P129E-G159D-Q245V-N248E-E271F,G020K-S024F-S078N-G118R-S128D-T213A-L217E, T022L-S024F-T213A-L217E,T022A-S024F-S101G-S103A-V104I-N116L-G118V-Y209A-A232V,N062E-S101A-S103A-V104I-S188D-A232V-Q245R,S024R-T033S-S128I-A158E-S188D, S024R-S078D-G118S-S188D-Q245R,T022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245R-N248D,G020R-S024R-S078G-G118D-G159D-S188D-H249R,S024R-S078G-G118D-P129E-G159D-Q245R-H249R,S024R-N062E-S078G-G118D-P129E-G159D-S188D-N248D-H249R,S024R-T033S-A158E-S188D, T022A-S101G-S103A-V104I-L148I,N116L-G118R-S128I-S166D, S024R-N062E-S078D-G118S-S188D-N248D,S078G-G118S-P129E-H249R, S101A-S103A-V104I-S188D-P210I-Q245R,T022A-S101G-S103A-V104I-N116L-G118V-Y209V-T213A-A232V,G020K-T022L-S024F-S078N-G118R-S166D, T022A-G118R-S128I-S188D,G020R-N062E-S078G-G118S-N248D, G020R-S078D-G118D-S188D-H249R,N062E-S078G-G118D, G020K-T022L-G118R-S166D-T213A,G061E-S101G-S103A-V104I-N116A-Y209V-G211Q-T213A-A232V,T022A-S101G-S103A-V104I-G159D-S188E-A232S-Q245V-N248E-E271L,T022A-S024F-T033S-S101G-S103A-V104I-N116A-Y209V-T213A-A232V,T022A-S024R-N076D-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-N269D-A270V-E271F-A272V,T022A-V068A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S101G-S103A-V104I-G159D-S188E-A232L-Q245T-N248E-E271T,S078G-G118S-P129E-S188D-N248D-H249R, G020K-T022L-S166D-L217E,T213A-L217E,T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245V-N248D-E271T,T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245R-N248D,T022A-S101G-S103A-V104I-G159D-S188E-A232L-Q245V-N248H,G020R-S078D-G118S-G159D-Q245R-N248D-H249R,S024F-S101G-S103A-V104I-N116A-Y209V-A232V,T022L-S024F-G118R-S166D-L217E, S024R-T033S-S188D,G020R-N062E-S078G-G118D-G159D-S188D-H249R-A272D, T033S-A158E-S188D,S101G-S103A-V104I-N116L-S128N-Y209A-G211Q-A232V,T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245T-N248E,G020R-S078D-G118D-G159D-Q245R,A016S-S024F-T033S-S101G-S103A-V104I-N116L-Y209V-G211Q-A232V,N062E-S078G-G118S-S188D, G020R-S024R-S078D-G118S-P129E-S188D-Q245R,A016S-S101G-S128N-L148I, G118R-S128I-S166D-S188D,N062E-S078G-G118D-P129E-G159D-H249R, T022A-T033S-N062E-N116L,N062E-S078G-G118D-P129E-Q245R,T022A-S024F-T033S-S101G-S103A-V104I-N116L-Y209V-T213A-A232V,T022A-S101G-S103A-V104I-G159D-S188D-A232T-Q245T-N248E-E271L,S078G-G118S-S188D-H249R, S024R-N062E-S078D-G118S-H249R,S078G-G118S-P129E-S188D-N248D,S101G-S103A-V104I-N116L-S128N-Y209V-T213A-A232V,T022A-S024R-N062E-N116L-G118R-A158E-S188D,G020K-S024F-S078N-S166D-L217E, S024R-S078G-G118S-P129E-H249R,T022A-S024R-S128I-A158E-S166D,S101G-S103A-V104I-N116L-Y209V-G211Q-T213A-A232V,T022A-S024F-T033S-S101G-S103A-V104I-N116L-Y209A-G211Q-A232V,G020R-S078D-G118D-P129E-G159D-S188D-Q245R,N062E-S078G-G118S-P129E-G159D-N248D-H249R, A016S-V104I-S128N-L148I,T022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245V-N248E,T022A-G023A-T033S-S101G-S103A-V104I-N116A-Y209A-G211Q-T213A-A232V,T022A-N116L-S188D, G020R-N062E-S078D-G118D-P129E-G159D-S188D-Q245R,S024R-N062E-S078D-G118D-P129E-H249R,S024F-T033S-S101G-S103A-V104I-N116L-Y209V-T213A-A232V,S024R-S078G-G118S-G159D-S188D-N248D,T022A-S024F-S101G-S103A-V104I-N116L-Y209V-A232V,T022A-S101G-S103A-V104I-N116L-S128N-Y209V-G211Q-A232V,S024R-N062E-S166D-S188D, S024R-S078D-G118S-G159D-S188D-Q245R-N248D,S101G-S103A-V104I-N116A-Y209V-T213A-A232V,T022A-S024R-G118R-A158E-S166D-S188D,T022A-T033S-S101G-S103A-V104I-N116L-Y209A-A232V,G020K-T022L-S024F-S078N-G118R-S128D,A016S-T022A-S024F-T033S-S101G-S103A-V104I-N116A-Y209V-T213A-A232V,S024R-N062E-N116L-S128I,T022A-S024F-S101G-S103A-V104I-N116L-Y209A-T213A-A232V,T022A-V068A-S101G-S103A-V104I-N116L-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S024R-T033S-N062E-G118R-A158E,S024R-N062E-S078D-G118D-P129E-Q245R-N248D-H249R,T022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245R-N248D,T022A-S024R-A158E-S188D, G020K-T022L-S078N-G118R-S166D-L217E,G020R-S078D-G118D-P129E,A016S-S101G-S103A-V104I-N116A-S128N-Y209V-G211Q-A232V,G020K-T022L-S024F-G118R-S166D-T213A-L217E, G118R-T213A-L217E,T022L-S024F-S166D-T213A-L217E,S024R-N076D-S101A-S103A-V104I-S188D-A232V-Q245R,T022A-T033S-G118R-P129E, S024R-S078G-G118S-P129E-G159D-S188D-N248D,T022A-S024R-S101G-S103A-V104I-I107V-P129E-G159D-S188D-A232V-Q245R-N248D-A270V-E271F,T022A-S101G-S103A-V104I-N116L-Y209V-G211Q-T213A-A232V,T022A-S101A-S103G-V104I-S128N, T022A-S024R-N062E-N116L-G118R-S166D,A016S-T022A-S024F-S101G-S103A-V104I-N116L-Y209A-T213A-A232V,A016S-S101G-S103A-V164I-N116L-Y209V-G211Q-T213A-A232V,S024F-G118R-S128D-T213A-L217E, T022L-S024F-G118R-T213A-L217E,N062E-S078D-G118S, S024R-G118R-A158E-S188D,S024F-S101G-S103A-V104I-N116L-S128N-Y209A-A232V, A088L, E271W,N062E-S078D-G118D-P129E-H249R, T022A-V104I-S128N-L148I,T022Q-S101G-S103A-V104I-G159D-S188E-A230V-A232V-Q245R-N248H,S101G-S103A-V104I-N116L-G118V-Y209A-T213A-A232V,S024R-S078G-G118D-G159D-Q245R-H249R, G097P-A098F-S099A-V104I,T022L-S024F-S078N-G118R-S166D-T213A,N062E-S078G-G118D-S188D-Q245R-N248D, T022A-S024R-T033S-S166D-S188D,T022A-T033S-R045C-G118R-A158E-S188D, T022A-S024R-N062E-S128I-S188D,T022A-S101G-S103A-V104I-P129E-G159D-S188D-A232V-Q245R-N248D-A270V-E271F,T022A-S024R-N116L-S166D, S024R-N062E-S128I-A158E,S024R-G118R-A158E-S166D, G020K-T022L-S078N-T213A,G020R-S024R-S078G-G118D-G159D-Q245R-N248D,G020K-T022L-S078N-T213A-L217E, G020R-S078D-G118D-G159D-Q245R-H249R,T022A-S024F-S101G-S103A-V104I-N116L-S128N-Y209A-G211Q-T213A-A232V,S024R-S101A-S103A-V104I-S188D-A232V-Q245R,S024R-N116L-G118R-A158E-S166D-S188D, S024F-S166D-T213A,G020R-S024R-N062E-S078D-G118S-P129E-Q245R-N248D-H249R,T022A-S024R-S128I-S166D-S188D,G020R-S024R-S078D-G118S-P129E-N248D-H249R,T022A-S024R-T033S-N116L-G118R-A158E-S166D-S188D,T022A-S024F-T033S-S101G-S103A-V104I-N116L-S128N-Y209A-T213A-A232V,T022L-S024F-L217E, G020R-S078G-G118S-G159D-S188D-Q245R-N248D-H249R,S099T-S103G, S024R-S078D-G118D-P129E-N248D-H249R,S024R-S078D-G118D-P129E-S188D-Q245R-N248D,T022A-T033S-S101G-S103A-V104I-G118R-G159D-S188D-T213A-A232V-Q245R-N248D-E271F,S024R-S078D-G118S-P129E-G159D-S188D-N248D-H249R,S078D-G118D-P129E-G159D-Q245R-H249R,A016S-T022A-S024F-S101G-S103A-V104I-N116A-Y209A-A232V,S024R-S078D-G118D-P129E-G159D-S188D-Q245R-N248D-H249R,T022A-S101G-S103A-V104I-G118R-S128L-G159D-S188D-T213A-A232V-Q245R-N248D-E271F,G020R-S078G-G118S-G159D-Q245R-N248D, S078G-G118D-G159D-S188D-Q245R,T022L-G118R-T213A-L217E, G020R-S024R-S078G-G118S-S188D-N248D-H249R,G118R-A158E-S166D-S188D,A016S-T022A-S024F-T033S-S101G-S103A-V104I-N116L-Y209V-T213A-A232V,N062E-S078G-G118S-P129E-G159D-Q245R-N248D,T022Q-A088S-S101G-S103A-V104I-G159D-S188D-A232T-Q245V-N248E,S078D-G118S-P129E-G159D-Q245R-N248D, G020K-S024F-S128D-N204K,S024F-S101G-S103A-V104I-N116A-Y209V-G211Q-A232V,S024R-S078D-G118S-S188D, S101G-S103A-V104I-N116L-Y209V-A232V,G020R-N062E-S078D-G118S-S188D-Q245R-N248D-H249R,A016S-T022A-S024F-S101G-S103A-V104I-N116L-Y209A-G211Q-A232V,S101G-S103A-V104L-L148I,T022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245T-N248D-E271L,S024R-N062E-S078D-G118D-P129E-S188D-H249R, R010H-T022L-S024F-L217E,T022A-S101G-S103A-V104I-G159D-S188E-A232L-Q245R-N248D,S024R-N062E-S078G-G118D-S188D-Q245R, S024R-N116L-S128I-A158E-S166D,T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245T-N248H-E271F,G020R-S078D-G118S-P129E-G159D,G020R-S024R-S078D-G118D-P129E-Q245R-N248D,S024R-N076D-S101A-S103A-V104I-P210I-A232V-Q245R,S078D-G118D-S188D-Q245R, G020K-T022L-S024F-G118R-S166D-T213A,S078G-G118S-G159D-Q245R-N248D-H249R,T022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245R-N248E-E271F,S056R-G065P, S056K,T022A-S024R-N076D-S101G-S103A-V104I-P129E-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S024R-N062E-N116L-G118R-S128I-A158E,S024R-S078G-G118S-P129E-S188D,G020R-N062E-S078D-G118D-G159D-N248D-H249R,S024R-S078G-G118S-G159D-N248D, S024R-N062E-S078G-G118D-S188D-N248D,G020R-S024R-N062E-S078D-G118S-P129E-Q245R, S024R-S078D-G118D-G159D,T022A-S024R-T033S-G118R-S128I-S166D-S188D,A016S-T033S-S101G-S103A-V104I-N116L-S128N-Y209V-A232V,T022A-T033S-S188D, G097P-S103A-V104L, G020R-S078G-G118D-P129E,N062E-S101A-S103A-V104I-S188D-V203L-A232V-Q245R,G020R-S024R-S078G-G118S-G159D-Q245R-N248D,G020R-S078D-G118D-G159D-S188D,G020R-S024R-S078G-G118D-S188D-Q245R-N248D,G020R-N062E-S078D-G118D-S188D, G020R-S101A-P210I-G211Q,T022A-T033S-N116L-S188D,T022A-T033S-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245R-N248E-E271L,S024R-N062E-S078D-G118D-N248D-H249R, T022A-S024R-N116L-A158E,N062E-N076D-S101A-S103A-V104I-S188D-P210I-A232V-Q245R,S024R-S078G-G118S-S188D-N248D-H249R,G020R-S078G-G118S-P129E-S188D-N248D-H249R,S024R-S078D-G118D-G159D-S188D,A016S-S101G-S103A-V104I-N116L-Y209A-G211Q-A232V, S132D, N043K,G097P-V104L,A016S-T022A-S024F-S101G-S103A-V104I-N116A-Y209A-G211Q-A232V,S024R-N062E-S078D-G118D-G159D-S188D-H249R,S024R-S078G-G118S-G159D-S188D, S024R-S078G-G118D-G159D-S188D-N248D,S024R-N062E-S078G-G118S-G159D-S188D-Q245R-H249R,S024F-S101G-S103A-V104I-N116A-Y209V-T213A-A232V,G020K-S078N-S166D-T213A-L217E, S103A-L111V-L148I,S024F-T033S-S101G-S103A-V104I-N116L-S128N-Y209A-G211Q-T213A-A232V,S024R-S078D-G118D-G159D-S188D-Q245R-N248D,T022A-T033S-S101G-S103A-V104I-N116A-Y209V-T213A-A232V,S024R-N062E-S078G-G118D-P129E-S188D,S024R-S078D-G118D-P129E-S188D-Q245R-H249R, T022A-G118R-S166D,G020R-S024R-S078G-G118S-G159D-S188D-Q245R,T022A-S024R-T033S-N116L-G118R-A158E-S188D,T022Q-S101G-S103A-V104I-G159D-S188E-A232T-Q245V-N248D-E271F,G020R-S024R-S078D-G118S-P129E-Q245R-N248D-H249R,A016S-T022A-V104I-S128N,S024R-N062E-N076D-S101A-S103A-V104I-S188D-P210I-A232V-Q245R,T022L-G118R-S166D-T213A-L217E,S078D-G118S-P129E-G159D-S188D-Q245R-H249R, G195W-N269E,G020R-S024R-S078G-G118S-P129E-Q245V-N248E, N269W,T022A-T033S-G118R-A158E-S166D-S188D,G020R-N062E-S078G-G118D-P129E-G159D-Q245R-H249R, A016S-S101A-S103A,G097P-S099G-S103A-V104I, S024R-S078D-G118D-P129E-Q245R,T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245R-N248E,S078G-G118D-P129E-S188D-Q245R, T033S-N062E-G118R-A158E,G020R-S078D-G118S-G159D-N248D, A016S-T022A-S103G-V104I-S128N-L148I,A016S-S101G-S103A-V104I-N116A-Y209A-A232V,S101A-S103A-V104I-S188D-M222S-A232V-Q245R,T022A-S024F-S101G-S103A-V104I-N116A-S128N-Y209V-T213A-A232V,A016S-T022A-S024F-S101G-S103A-V104I-N116A-Y209A-T213A-A232V,T022A-S101G-S103A-V104I-G159D-S188D-T213A-A232V-Q245R-N248D-E271F,A016S-S101G-S103A-V104I-N116L-Y209A-T213A-A232V,G020R-S078D-G118S-S188D, G020R-S078D-G118S-P129E-Q245R-N248D,T022L-S166D-T213A, G020R-S024R-S078D-G118S-P129E-G159D-S188D-N248D,S240G, S078G-G118S-P129E-Q245R-N248D,S024R-S078D-G118D-P129E-G159D-Q245R-N248D, S024R-S078G-G118S-G159D,T022A-S024R-N076D-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F-A272V,N062E-S078G-G118S-P129E-S188D-Q245R-N248D-H249R,S024F-S078N-G118R-S166D, S078G-G118S-P129E-N248D, N155W,A016S-S024F-S101G-S103A-V104I-N116A-S128N-Y209V-G211Q-T213A-A232V,G020R-N062E-S078D-G118D-P129E-Q245R-N248D,G020R-S078D-G118D-P129E-S188D-Q236R-N248D-H249R,T022A-S024R-N116L-A158E-S188D,S101G-S103A-V104I-N116A-S128N-Y209V-G211Q-T213A-A232V,S078D-G118S-S188D-Q245R-N248D, T022L-S024F-S078N-L217E,S099A-S101A-G102A, G020R-S078D-G118D-G159D-N248D-H249R,T022A-N076D-S101G-S103A-V104I-I107V-P129E-G159D-S188D-A232V-Q245R-N248D-E271F,T033S-N116L-G118R-S166D-S188D, S024R-G118R-S128I-A158E-S166D-S188D,G020R-S024R-N062E-S078G-G118S-P129E-G159D-Q245R-N248D-H249R,N062E-S078G-G118D-N248D-H249R, N062E-S078R-P129E-S188D-Q245R-N248D,S024R-N062E-G159D-S188D-Q245R, G020R-N062E-G118R-S188D-N248D-H249R,N062E-S078R-P129E-G159D, G020R-S024R-N062E-P129E-S188D-N248D,N062E-S078R-G118R-G159D-S188D-P210L-N248D, N062E-S078R-Q245R-N248D,G020R-G159D-S188D-H249R, G020R-G118R-G159D-S188D-N248D,G118R-P129E-G159D-S188D-H249R, N062E-S078R-G118R-P129E-N248D,N062E-S078R-G159D, N062E-G118R-P129E-S188D-Q245R,G020R-S024R-P129E-G159D-S188D-N248D, S024R-N062E-S078R-G159D-N248D,G020R-P129E-S188D-H249R, G020R-P129E-G159D-S188D-Q245R,A016S-S024F-S101G-S103A-V104I-N116A-S128N-S188D-Y209V-T213A-A232V,G020R-G159D-S188D-Q245R-N248D, G118R-P129E-G159D-Q245R-N248D,G020R-N062E-S078R-P129E-S188D-N248D, G020R-P129E-S188D-Q245R,S024R-N062E-S188D-N248D, S078R-L111I-N185I-I198L-L217E,G020R-P129E-S188D-N248D-H249R, G020R-N062E-G118R-G159D-N248D,G020K-T022L-G118R-P129E-S188D, G020R-G118R-P129E-S188D,G020R-P129E-G159D-Q245R-N248D, G020R-S024R-P129E-G159D,G020R-P129E-G159D-Q245R-N248D-H249R,S024R-P129E-G159D-S188D-Q245R-N248D-H249R, G020R-P129E-Q245R-N248D,N062E-S078R-G159D-N248D,S024R-G118R-P129E-G159D-S188D-Q245R-N248D-H249R,G020R-G061W-P129E-G159D-S188D-Q245R,T022A-S101G-S103A-V104I-G159D-S188D-M222S-A232V-Q245R-N248D-A270V-E271F,N062E-G159D-S188D-H249R,A016S-S024F-S101G-S103A-V104I-N116A-S128N-S130D-Y209V-T213A-A232V,G020R-S024R-P129E-G159D-N248D-H249R, P129E-G159D-S188D-H249R,G118R-P129E-G159D-S188D-K237R-N248D-H249R, G020R-G159D-S188D-Q245R,G020R-S188D-Q245R-N248D-H249R,T022A-S101G-S103A-V104I-P129S-L217Q-A232V-Q245R,G020R-S078R-G118R-P129E-S188D-N248D,G020R-S024R-G118R-G159D-S188D-Q245R-N248D,G020R-G118R-P129E-G159D-S188D, G020R-G159D-S188D,G020R-G118R-P129E-S188D-N248D,A016S-S024R-S101G-S103A-V104I-N116A-S128N-Y209V-T213A-A232V,G020K-N062E-S188D-T213A-M222S,A016S-S024F-S101D-S103A-V104I-N116A-S128N-Y209V-T213A-A232V,S024G-S078N-S101N-Q109G-S128A-L217Q,T022A-G097S-S101N-S103A-V104I-S128A-G159D-S188D-L217Q-M222S-A232V-Q245R-N248D-E271F,T022A-S101G-S103A-V104I-G159D-S188D-M222S-A232V-Q245R-N248D-E271F,G020R-P129E-Q245R, S024R-P129E-G159D-Q245R-N248D,S024R-G118R-S188D-N248D, G020R-P129E-H249R, S024R-P129E-Q245R-N248D,T022A-S101G-S103A-V104I-L217Q-A232V-Q245R,G020R-S078R-P129E-G159D-S188D-H249R,T022A-S101G-S103A-V104I-S128A-P129S-L217Q-A232V-Q245R,G118R-P129E-H249R, G020R-N062E-G118R,G020R-G118R-P129E-G159D-Q245R-N248D,T022A-S101Q-S103A-V104I-P129S-L217Q-A232V-Q245R,S024R-S078R-G118R-P129E-G159D-S188D, P129E-Q245R-N248D-H249R,S024G-G061S-S078N-S101N-Q109G-S128A-L217Q-N243V,S078R-V084A-P129E-Q245R, S024R-G118R-G159D-S188D-N248D,G020R-G118R-P129E-G159D-H249R, S078R-P129E-G159D,G061P-S078N-G097A-S101N-S128A-V203Y-L217Q,P086S-S087G-A088V-G097A-N117S-S128A-L217Q,S024R-S078R-G159D-S188D-Q245R-N248D-H249R, S078R-G118R-P129E,S024G-S078N-S101N-S128A-L217Q-N243V, S024R-S078R-G159D-S188D,G020R-G118R-P129E, G020R-S024R-P129E-G159D-S188D-H249R,S024R-G118R-G159D-S188D, S024R-G118R-P129E-G159D-S188D-Q245R-N248D,S024G-S078N-S101N-S128A-L217Q, S024R-G159D-S188D-Q245R,S024G-S078N-S101N-Q109G-L217Q-N243V-N248A,G020R-S078R-G159D-S188D-Q245R-N248D, S078R-G118R-P129E-S188D-N248D,S024R-S188D-Q245R-N248D, G020R-S024R-S101A-P210I-G211Q,G020R-G159D-N248D-H249R, G020R-P129E, G020K-N062E-N116L-T213A-M222S,S024G-S078N-A088T-S101N-Q109G-N116T-S128A-A158S-L217Q-N218S-L257G,A016S-S024F-S101G-S103A-V104I-N116A-S128N-S166D-Y209V-T213A-A232V,S024R-N062E-S078R-P129E-H249R,N018K-G020R-S024R-R045T-S101A-P210I-G211Q-T213A,G020R-S024R-G118R-P129E-G159D-S188D-N248D-H249R,G020R-S024R-P129E-H249R, G020R-G118R-S188D-Q245R-N248D-H249R,G020R-S024R-N062E-S078G-G118S-Q245R,G020R-S024R-G118R-P129E-G159D-N248D,S024G-S078N-S101N-Q109G-S128A-L217Q-S256R,S024G-S078N-S101N-S128A-A158S-L217Q-L257G,G020R-S024R-G118R-S188D-N248D,G020R-G118R-G159D-S188D-Q245R-N248D-H249R, G159D-S188D-Q245R-N248D,S078N-G118R-S166D-L217E-M222S,G020R-G118R-P129E-G159D-Q245R-N248D-H249R,T022A-S101Q-S103A-V104I-L124V-S128A-P129S-A232V-Q245R,S024R-N062E-Q245R, S188D-Q245R-N248D, S024R-G118R-P129E-S188D-H249R,T022A-G097A-S101Q-S103A-V104I-P129S-A232V-Q245R,G020R-G118R-S188D-Q245R-N248D,S024G-S078N-S101N-Q109G-N116T-S128A-L217Q-N243V-S256R, G020R-G118R,G020R-S024R-S188D, N062E-P129E-G159D-S188D-Q245R,G020R-G159D-Q245R-N248D, G020R-N043R-S101A-P210I-G211Q,G097A-S101N-S128A-L217Q, G020R-G118R-S188D-N248D, S024R-S188D-N248D,G020R-S024R-S188D-Q245R-N248D,G020R-S024R-G118R-P129E-G159D-N248D-H249R, G020R-P129E-N248D,G159D-S188D, P129E-G159D, G020R-S188D-H249R, P129E-G159D-Q245R-N248D,G020R-G118R-G159D-N248D-H249R, G118R-S188D-N248D-H249R,G020K-S101D-G102A-L217E-S240R, N043R-G102A-L217E,N062E-S078N-S101D-S240R, G102A-L217E, G020K-S128L-L217E-S240R,G020K-N062E-S078N-L217E-S240R, N043R-S101D-S128L-L217E-S240R,G020K-S078N-S101D-G102A-P210L, N043R-S101D-L217E,G020K-G100S-L217E-S240R, S078N-G100S, G020K-N043R-S101D-G102A-L217E,G100S-S240R, G020K-G102A-L217E,G020K-S024G-N062E-S078N-S101N-Q109N-N116L-S128A-S188D,G020K-L217E-S240R, N043R-S101D, N043R-S078N-S101D-S128L-S240R,N043R-S078N-S101D-L217E-S240R, G020K-S078N-S101D, N043R-S128L-S240R,G020K-N062E-N116L-N123G-T213A,G020K-N062E-G097S-S101G-Q109G-N116L-S128A,G020K-N062E-S078N-G097A-S101N-Q109N-N116L-S188D,N043R-S078N-S101D-G102A-L217E, G020K-S078N-S099G-S128L-L217E,G020K-N062E-S101D, G020K-S128L-L217E,G020K-S024G-N062E-S078N-G097S-S101G-Q109G-N116L-L217Q,N043R-G097A-S101D-S128L, G020K-S024G-N062E-S101Q-Q109N-N116L-S128A,G020K-S024G-N062E-S078N-S101N-N116L-S128A-L217Q, S078N-G102A-S240R,N043R-S078N-G100S-L217E-S240R, G020K-S078N-S128L-L217E-S240R,G020K-N062E-N116L-S128Q, G020K-S101G-L217E-S240R,G020K-S024G-N062E-S101N-Q109G-N116L-S128A-L217Q,G020K-N062E-S078N-G097S-S101N-N116L-L217Q,G020K-N062E-G097S-S101N-Q109G-N116L-L217Q,G020K-S024G-N062E-S078N-G097A-S101Q-Q109N-N116L-M222S,G020K-S024G-N062E-S078N-G097S-S101Q-Q109N-N116L-S28A-S188D-L217Q,G020K-N062E-S078N-S101N-Q109G-N116L-S188D-L217Q, orG020K-N062E-S105G-N116L-S128L-T213A (List 14), and wherein amino acidpositions of the subtilisin variant are numbered according to thenumbering of corresponding amino acid positions in the amino acidsequence of Bacillus amyloliquefaciens subtilisin BPN′ shown in SEQ IDNO:1.

The present invention further provides an isolated subtilisin variant,wherein said subtilisin variant is a mature form having proteolyticactivity and comprises an amino acid sequence comprising a combinationof amino acid substitutions at positions selected fromT022A-S101G-S103A-V104I-N116L-G159D-S188D-A232V-Q245R-N248D,S024R-N116L-A158E-S188D, G020K-S024F-S078N-G118R-T213A,T022A-S024R-T033S-N116L-G118R-S128I, G020R-S078D-G118D-S188D-Q245R,S024F-S101G-S103A-V104I-N116A-Y209A-T213A-A232V,S024R-N062E-S078G-G118D-G159D-S188D,T022A-N062E-N116L-G118R-A158E-S166D, G020K-T022L-S078N-S166D-L217E,G020R-S078D-G118D-G159D, T022A-S024R-T033S-N116L,G020R-S078D-G118D-P129E-G159D, G020R-S078D-G118D-G159D-H249R,S024R-N062E-S078G-G118S, S024R-S078D-G118D-S188D-Q245R-H249R,A016S-S024F-T033S-S101G-S103A-V104I-N116L-Y209V-T213A-A232V,G020R-N062E-S078D-G118D-P129E-G159D-Q245R,S024F-S101G-S103A-V104I-N116L-G118V-Y209V-G211Q-A232V,S024F-S166D-L217E, T022A-S024R-S166D,S024R-N062E-S078G-G118D-P129E-N248D,A016S-S101G-S103A-V104I-N116L-S128N-Y209V-A232V,A016S-T022A-S101G-S103A-V104I-N116A-Y209V-G211Q-A232V,S078D-G118S-G159D-N248D-H249R, S024R-S078G-G118D-P129E-G159D-S188D,T022A-S024R-N062E-N116L-G118R-S128I-S188D, S103A-S128N,T022L-S078N-G118R-S166D-T213A,S024F-T033S-S101G-S103A-V104I-N116L-S128N-Y209A-T213A-A232V,T022A-S024R-N076D-S101G-S103A-V104I-P129E-G159D-S188D-A232V-Q245R-N248D-E271F-A272V,S024R-S078D-G118S-S128N-P129E,S024R-S078G-G118S-P129E-G159D-S188D-Q245R-H249R,T022A-S024R-N076D-S101G-S103A-V104I-P129E-G159D-S188D-A232V-Q245R-N248D-A270V-E271F-A272V,T022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245T-N248D,S024R-T033S-N116L-A158E, S130E-N269K, S024R-T033S-N116L-S128I-A158E,A016V-G020R-S024R-S078D-G118D-P129E-Q245R-N248D,S099G-S101A-G102A-S103G-V104I, A016S-T022A-S101A-V104L-S128N-L148I,G020R-S024R-S078G-G118S-P129E-S188D, S259W, S259H,T022A-S101G-S103A-V104I-P129E-G159D-S188D-A232V-Q245R-N248D-E271F,N018D-S101G-S103A-V104I-N116A-Y209A-G211Q-T213A-A232V,G020K-S024F-S078N-S166D-T213A,N076D-S101A-S103A-V104I-S188D-A232V-Q245R,S078D-G118D-P129E-G159D-H249R, S024R-S078G-G118D-P129E-G159D-N248D,S024R-N062E-S101A-S103A-V104I-S188D-A232V-Q245R, E271R,G020R-S024R-S078D-G118D-P129E-G159D-S188D,T022A-S024R-N076D-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-A270V-E271F,G020R-N062E-S078D-G118S-G159D-S188D-Q245R-H249R,Q012R-S101G-S103A-V104I-N116L-S128N-Y209V-A232V,G020R-S078G-G118D-P129E-G159D-S188D-Q245R-N248D,G020R-N062E-S078G-G118S-P129E-G159D-S188D-Q245R-H249R,T022A-S101G-S103A-V104I-G118R-S128L-G159D-S188D-A232V-Q245R-N248D-E271F,G020R-S078D-G118D-G159D-S188D-Q245R-N248D,T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F-A272V,G020R-S024R-N062E-S078D-G118S-P129E-S188D-Q245R-N248D-H249R,G020R-S078G-G118S-G159D-H249R, G020K-T022L-S024F-G118R-S128D-S166D,T022A-S024R-N062E-N116L-G118R-S128I-A158E-S166D, A016S-S103G-V104L,A016S-T033S-S101G-S103A-V104I-N116L-Y209A-A232V,G020R-S024R-N062E-S078D-G118S-P129E-S188D-N248D,G020K-T022L-S078N-G118R-S166D, G020K-T213A-L217E,S024R-N062E-S078D-G118S-P129E-G159D-S188D-Q245R-N248D-H249R,S078N-S166D-T213A-L217E, S078D-G118S-P129E-G159D-Q245R-H249R,T022A-N076D-S101G-S103A-V104I-P129E-G159D-S188D-A232V-Q245R-N248D-E271F-A272V,G020K-T022L-G118R-L217E, T022A-V104L-L111V,T022A-S101G-S103A-V104I-S128A-P129E-G159D-S188D-A232V-Q245R-N248D-E271F,S024R-N062E-S078G-G118S-P129E-S188D-N248D,G020R-S024R-S078D-G118D-G159D-S188D-N248D, T022L-S078N-L217E,G020R-S024R-S078G-G118S-S188D,S024F-S101G-S103A-V104I-N116L-Y209V-G211Q-T213A-A232V,T022A-N116L-G118R-A158E-S166D,G020R-S024R-S078G-G118S-P129E-S188D-Q245R-N248D-H249R,T022A-V104I-S128N, S024R-S078G-G118D-Q245R,N062E-S078G-G118S-P129E-G159D-S188D-H249R,T022A-S101G-S103A-V104I-I107V-G159D-S188D-A232V-Q245R-N248D-A270V-E271F-A272V,S024R-N062E-S078D-G118S-P129E-G159D-Q245R-N248D-H249R,T022A-T033S-G118R-A158E-S188D,T022A-S078N-G097A-S101N-S103A-V104I-S128A-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-S024R-S166D-S188D,A016S-S024F-T033S-S101G-S103A-V104I-N116L-Y209A-A232V,T022A-T033S-S128I-A158E, S024R-T033S-N116L-S188D,N062E-N076D-S101A-S103A-V104I-P210I-A232V-Q245R,T022A-N076D-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-A270V-E271F-A272V,S130D, G020K-T022L-S024F-S078N-G118R-S166D-L217E,T022Q-S101G-S103A-V104I-G159D-S188E-A232S-Q245V-N248E,S024R-T033S-N116L-S166D-S188D, S024R-S078D-G118S-S188D-Q245R-H249R,T022A-S024R-T033S-A158E-S166D-S188D,G020K-T022L-S024F-S166D-T213A-L217E, T022A-N062E-N116L-G118R-S188D,G020R-S078G-G118D-Q245R-N248D-H249R, T022A-N116L-G118R-S128I,T022L-S078N-S166D, T022L-G118R-S128D, G097P-S099A,T022L-S024F-G118R-S128D, S242K, N062E-S078D-G118S-G159D,A016S-S103G-S128N-L148I, G097P-A098Q-S101A,T022A-S103A-V104L-S128N-L148I,T022A-S101G-S103A-V104I-G118R-G159D-S188D-T213A-A232V-Q245R-N248D-E271F,S099G-S101G-G102A,T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245T-N248D,G100S-S101G-V104I, S078D-G118S-G159D-Q245R-N248D,A016S-S024F-T033S-S101G-S103A-V104I-N116A-Y209V-G211Q-T213A-A232V,T022A-S024R-T033S-N116L-A158E,T022A-N076D-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F-A272V,T022A-S024R-N116L-S128I-S166D,S101G-S103A-V104I-N116L-S128N-Y209V-A232V,S024F-S101G-S103A-V104I-N116L-Y209A-A232V,G020R-S078G-G118D-G159D-S188D-N248D,G020R-S078D-G118D-S188D-Q245R-N248D, S024F-S078N-G118R-S128D,T022L-S024F-S078N-G118R-S166D,G020R-S024R-S078D-G118S-P129E-G159D-S188D-Q245R-H249R,S103A-V104L-S128N, T022A-S024R-N062E-N116L-S128I,T022A-N062E-N116L-G118R-A158E-S188D,T022A-S024G-G097S-S101G-S103A-V104I-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,G020R-S024R-N062E-S078D-G118D-G159D-S188D-Q245R-H249R,N062E-S078D-G118S-S188D-Q245R-N248D, T022A-S103A-L148I,T022A-S024R-N116L-S128I-A158E-S188D, G097P-S099T-S101G-V101I,S166D-T213A-L217E, A016S-T033S-S101G-S103A-V104I-N116L-Y209V-A232V,N062E-S078D-G118S-P129E-G159D-S188D-Q245R,T022A-S024F-S101G-S103A-V104I-N116A-S128N-Y209A-G211Q-A232V,G020K-T022L-S078N-G118R-S128D-S166D-L217E, T033S-S188D,T033S-N116L-G118R-A158E,T022A-S024G-N076D-V093I-S101N-S103A-V104I-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,G020R-S078D-G118S-P129E-Q245R-H249R, T022A-S024R-A158E-S166D-S188D,G020K-T022L-S078N-G118R-S128D, G020R-S024R-S078D-G118D-P129E-S188D,S099T-G102A, S024R-N116L-G118R-A158E-S188D, T033S-N116L-G118R-S166D,T022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245V-N248H,S078D-G118D-Q245R, G020K-S078N-G118R-S166D-T213A-L217E-A272D,A016S-T022A-V104L-L111V-L148I,T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245V-N248H,N062E-N116L-G118R-S166D, N062E-S166D,T022A-S024R-N062E-G118R-A158E-S188D,T022A-S024F-S101G-S103A-V104I-N116A-Y209V-A232V,T022A-S024R-N076D-S101G-S103A-V104I-P129E-G159D-S188D-A232V-Q245R-N248D-A270V-E271F,G020R-S024R-N062E-S078G-G118D-Q245R, S024R-N116L-S128I-A158E-S188D,S024R-N062E-S101A-S103A-V104I-P210I-A232V-Q245R, A114T,T022L-S024F-S078N-G118R-S128D, S024R-N116L-G118R-S166D,N062E-S078G-G118D-P129E,A016S-T022A-S101G-S103A-V104I-N116L-Y209A-G211Q-A232V,G020K-T022L-S078N-S166D,G020R-S024R-N062E-S078G-G118S-P129E-S188D-Q245R-N248D-H249R,T022A-S078N-G097A-S101Q-S103A-V104I-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-S101G-S103G,A016S-S024F-S101G-S103A-V104I-N116L-S128N-Y209A-G211Q-S212L-A232V,S024F-G118R-S166D-T213A, G100S-S101G, A016S-T022A-S103A-V104L-S128N,G020K-S024F-S078N-G118R-S166D, S099T-G102A-S103G,G020K-S024F-S128D-L217E, S078D-G118D-G159D-H249R,N062E-S078G-G118D-S188D, N076D-S101A-S103A-V104I-P210I-A232V-Q245R,S024R-S078G-G118D-P129E-N248D, S078G-G118S-G159D-S188D,A016S-S101G-S103A-V104I-N116L-S128N-Y209V-G211Q-T213A-A232V,G020K-S078N-G118R-S166D-T213A-L217E,T022A-S101N-S103A-V104I-L124V-P129S-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S101N-S103A-V104I-P129S-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,S024R-S078D-G118D-P129E, G020K-S024F-S128D-T213A,T022A-T033S-A158E-S188D, G020K-S024F-S128D-S166D-T274A,S024R-N062E-N116L-G118R-S128I-A158E-S166D-S188D,S078G-G118S-P129E-S188D, T022A-S024R-S128I-A158E-S188D,T022A-S024R-G118R-S128I-A158E, T022A-N116L-S166D,G020K-G118R-S128D-S166D, G020K-T022L-S024F-S078N-G118R-S128D-L217E,G020R-S024R-S078D-G118D-P129E-S188D-N248D,G020K-T022L-G118R-S128D-L217E, S024F-S078N-G118R-S128D-T213A,S024R-S078G-G118D-S188D-N248D,N062E-N076D-S101A-S103A-V104I-S188D-A232V-Q245R,T033S-G118R-S128I-A158E-S166D,A016S-S101G-S103A-V104I-N116A-S128N-Y209V-A232V,S101G-V104I-L111V-S128N,T022A-S078N-G097S-S101N-S103A-V104I-S128A-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245V-N248E,T022A-S078N-G097A-S101N-S103A-V104I-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,G020K-T022L-S024F-G118R-S128D,T022A-N076D-S078N-G097S-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S024R-S101G-S103A-V104I-P129E-G159D-S188D-A232V-Q245R-N248D-A270V-E271F,T022A-S101G-S103A-V104I-P131L-G159D-S188D-A232V-Q245R-N248D-E271F,T022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245T-N248E,T022A-N076D-S078N-S101N-S103A-V104I-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S101G-S103A-V104I-N116L-S128N-Y209V-T213A-A232V,T022A-S024R-T033S-N062E-G118R-A158E-S188D,T022A-S024R-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-N116L-G118R-S188D,T022A-G097A-S101Q-S103A-V104I-L124V-S128A-P129S-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S101G-S103A-V104I-N116L-Y209V-T213A-A232V, T022A-S128I-S188D,G020R-S024R-S078G-G118D-G159D, S024R-T033S-N116L-S128I-A158E-S188D,T022A-T033S-S101G-S103A-V104I-G159D-S188D-T213A-A232V-Q245R-N248D-E271F,A016S-T033S-S101G-S103A-V104I-N116L-S128N-Y209A-A232V,A016S-S101G-S103G-V104I-L148I, S024R-T033S-G118R-S166D-S188D,T022A-T033S-S101G-S103A-V104I-N116L-Y209V-T213A-A232V,T022A-N062E-N116L-S128I, A016S-S101G-S103G-V104L-L148I,T022A-N116L-S166D-S188D, G020K-T022L-S024F-S078N-S128D-T213A,S099G-G100S-V104L, S078G-G118D-P129E-G159D-S188D-H249R-T253M,A016S-T022A-S101G-S103A-V104I-N116A-S128N-Y209A-A232V,T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245T-N248E,G020R-S078D-G118D-P129E-G159D-S188D-N248D-H249R,G118R-S166D-T213A-L217E, G020K-S024F-S128D,T022A-S024F-S101G-S103A-V104I-N116L-S128N-Y209V-G211Q-T213A-A232V,T022A-G097S-S101Q-S103A-V104I-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,G020K-T022L-G118R-S128D-T213A-L217E, S024F-G118R-S128D-T213A,T022A-S101A-S103A-V104I-S128N,S024R-N062E-N076D-S101A-S103A-V104I-S188D-A232V-Q245R,T022A-S103A-S128N-L148I, G020R-N062E-S078G-G118D-G159D-S188D-N248D,T022A-S024R-T033S-G118R-S128I-S188D,T022A-S101G-S103A-V104I-S128L-G159D-S188D-A232V-Q245R-N248D-E271F,S101G-S103A-V104I-N116L-S128N-Y209V-G211Q-A232V,T022A-S101Q-S103A-V104I-P129E-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-S024G-S078N-G097A-S101N-S103A-V104I-S128A-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-G097A-S101N-S103A-V104I-P129S-G159D-S188D-A232V-Q245R-N248D-E271F,T033S-S166D-S188D, S078D-G118S-P129E-Q245R-N248D,G020K-S024F-S078N-G118R-S166D-T213A,S024R-N062E-N116L-G118R-S166D-S188D,G020R-N062E-S078D-G118D-P129E-G159D-N248D-H249R,G020K-S078N-G118R-S128D, T022A-N062E-S166D, P014R, S240D, N155G,A016S-T022A-S024F-S101G-S103A-V104I-N116A-S128N-Y209A-T213A-A232V,A016S-T022A-T033S-S101G-S103A-V104I-N116L-Y209V-G211Q-A232V,A016S-T022A-S024F-T033S-S101G-S103A-V104I-N116L-Y209A-G211Q-T213A-A232V,T022A-G097S-S101N-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,A016S-S101G-S103A-V104I-N116L-S128N-Y209V-G211Q-A232V,G118R-S128I-S166D,T022A-S078N-G097S-S101N-S103A-V104I-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,S024R-A158E-S188D,T022A-S101N-S103A-V104I-L124V-P129E-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,N116L-G118R-S188D,T022A-G097A-S101N-S103A-V104I-L124V-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-G118R-S128I-A158E, G097P-S101A-V104L, S103G-V104I-S128N-L148I,G020R-N062E-S078G-G118D-P129E-S188D, V026W,G020R-N062E-S078D-G118D-P129E-G159D,T022A-N076D-S101G-S103A-V104I-I107V-N123S-P129E-G159D-S188D-A232V-Q245R-N248D-E271F-A272V,S103G-S128N-L148I,T022A-S024R-N076D-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S024G-N076D-S078N-S101G-S103A-V104I-S128A-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-S078N-G097S-S101G-S103A-V104I-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-N076D-S101N-S103A-V104I-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,N062E-S078D-G118S-G159D-S188D-Q245R-H249R,S024R-N076D-S101A-S103A-V104I-A232V-Q245R, T022L-S078N,A016S-T022A-S024F-S101G-S103A-V104I-N116L-Y209V-T213A-A232V,G020K-T022L-S128D-T213A, N062E-G118R-S128I-S188D,S024R-N116L-S128I-S166D-S188D,T022A-S101N-S103A-V104I-S128A-P129E-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,S024R-T033S-N116L-G118R-A158E,T022A-N076D-G097A-S101Q-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,T022L-S024F-S166D-L217E, S024R-S078D-G118S-G159D-N248D-H249R,T022A-S101Q-S103A-V104I-P129S-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-N076D-S101G-S103A-V104I-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,A016S-T022A-S024F-S101G-S103A-V104I-N116A-S128N-Y209A-A232V,S078G-G118D-P129E-Q245R-N248D, G020R-S078G-G118D-G159D-N248D,G020K-T022L-G118R-S128D-S166D,T022A-S024G-S101Q-S103A-V104I-S128A-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-S024G-N076D-S078N-G097A-S101G-S103A-V104I-P129E-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245T-N248D,G020R-N062E-S078G-G118D-S188D-Q245R-H249R,S024F-S101G-S103A-V104I-N116L-Y209A-T213A-A232V,S024R-N062E-N076D-S101A-S103A-V104I-A232V-Q245R,T022A-S024R-N116L-G118R-S128I-A158E, A098Q-S099T-S101A-V104L,T022A-S024G-G097S-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S078N-S101G-S103A-V104I-S128A-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-S101G-S103A-V104I-P129E-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,S099G-G102A,T022A-N076D-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-A270V-E271F,T022A-S078N-G097S-S101N-S103A-V104I-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-N076D-S101G-S103A-V104I-G159E-S188D-A232V-Q245R-N248D-E271F,T022A-G097A-S101G-S103A-V104I-P129E-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-N076D-S101N-S103A-V104I-S128A-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F-T274A,A016S-V104I-L148I, G097P-S101A-S103A, G020K-T022L-S024F-G118R-S166D,T022A-S024R-N062E-N116L-G118R-S128I-S166D,A016S-S024F-T033S-S101G-S103A-V104I-N116A-Y209A-G211Q-T213A-A232V,T022A-S101Q-S103A-V104I-L124V-P129E-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-N076D-S101G-S103A-V104I-S128A-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-S024G-N076D-S078N-S101G-S103A-V104I-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,N062E-S078G-G118S-P129E-N248D, G020R-S078D-G118S,T022A-S101Q-S103A-V104I-L124V-S128A-P129E-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,G097P-S101G-S103A-V104I, G020K-T022L-S166D-T213A-L217E,T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-A270V-E271F,T022A-S078N-S101N-S103A-V104I-P129E-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,A016S-S101A-S103A-S128N,T022A-S101G-S103A-V104I-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,A098Q-S099T-G102A-S103A,T022A-N076D-S078N-G097A-S101G-S103A-V104I-S128A-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,S078N-G118R-H120N-S128D-S166D, S101G-S103G-L148I,T022A-S024R-T033S-A158E-S166D, N116L-G118R-S166D,T022A-N062E-N116L-S166D,N076D-S101A-S103A-V104I-S188D-P210I-M222S-A232V-Q245R,T022A-S101G-V104L-L148I,T022A-S101G-S103A-V104I-G159D-S188E-Q245V-N248D,S024R-N076D-S101A-S103A-V104I-S188D-P210I-M222S-A232V-Q245R,T022A-S024G-N076D-G097S-S101N-S103A-V104I-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,N116L-G118R,T022A-S024G-S101N-S103A-V104I-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T033S-S101G-S103A-V104I-N116L-S128N-Y209A-G211Q-T213A-A232V,T022L-G118R-S166D-T213A,A016S-T022A-S101G-S103A-V104I-N116A-Y209V-A232V,T022A-N076D-S101G-S103A-V104I-P129E-G159D-S188D-A232V-Q245R-N248D-A270V-E271F,T022A-S101G-S103G-V104L-L148I, S024R-S078G-G118D-S188D,T022A-S101G-S103A-V104I-G159D-S188D-A232V-N238Y-Q245R-N248D-A270V-E271F-A272V,T022A-S024G-S078N-S101Q-S103A-V104I-G159D-S188D-S216G-L217Q-A232V-Q245R-N248D-E271F,S024R-N116L-S128I, P086T-G097P-S099T-S103A-V104I, 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G020R-S078G-G118S-P129E-Q245R-H249R,A016S-S024F-S101G-S103A-V104I-N116A-S128N-Y209V-T213A-A232V,T022A-S078N-S101Q-S103A-V104I-R145K-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-N043R-N076D-S101G-S103A-V104I-P129E-A158E-S188D-A232V-Q245R-N248D-E271F,T022A-G097A-S101Q-S103A-V104I-L126I-P129S-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-G097A-S101N-S103A-V104I-Q109N-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S024G-S078N-S101N-S103A-V104I-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S024R-N076D-S101G-S103A-V104I-P129E-S188D-A232V-Q245R-N248D-E271F-A272V,T022L-S078N-T213A,T022A-N076D-S078N-G097A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S078N-S101G-S103A-V104I-Q109G-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,N062E-S078G-G118S-P129E-G159D-Q245R-H249R,A016S-S101G-V104I-S128N-L148I, T022A-G118R-S188D,T022A-G097A-S101Q-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,G020K-S024F-G118R-S128D-T213A,T022A-S101Q-S103A-V104I-P129E-G159D-S188D-A232V-Q245R-N248D-E271F,G020K-T022L-S024F-S078N-S128D-T213A-L217E,T022A-S024G-S078N-G097A-S101Q-S103A-V104I-Q109G-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,A016S-S101G-S103A-V104I-N116L-Y209V-G211Q-A232V,T022A-S024G-G097A-S101Q-S103A-V104I-S128A-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-S101N-S103A-V104I-L124V-S128A-P129S-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,S099G-G100S-S103A,T022Q-S101G-S103A-V104I-S166D-S188E-A232T-Q245T-N248E,T022A-S024G-N076D-S078N-G097S-S101N-S103A-V104I-Q109N-G159D-S188D-A232V-Q245R-N248D-E271F,A098F-S099T-S103A-V104I,G020K-T022L-N062E-S078D-G118D-P129E-Q245R-N248D,T022L-S078N-G118R-S128D,T022A-S101G-S103A-V104I-N116L-Y209A-G211Q-T213A-A232V,T022A-S101Q-S103A-V104I-L126I-S128A-P129S-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,G023A-S024F-S101G-S103A-V104I-N116A-Y209V-A232V,T022A-S024R-N116L-G118R-S166D,S024R-N062E-S078D-G118D-P129E-G159D-Q245R-N248D-H249R,G020K-T022L-S024F-S078N-G118R-S128D-T213A,T022A-S024G-N076D-S078N-G097A-S101Q-S103A-V104I-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S024G-S101N-S103A-V104I-S128A-G159D-S188D-A232V-Q245R-N248D-E271F-T274I,G097P-S099A-V104L, G097P-S099A-S103A,T022A-S024G-S078N-G097S-S101Q-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-V104L-L148I,T022A-S024G-G097A-S101N-S103A-V104I-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S024R-S101G-S103A-V104I-P129E-G159S-S188D-A232V-Q245R-N248D-E271F,T022A-S024G-N076D-S078N-S101G-S103A-V104I-Q109G-G159D-S188D-A232V-Q245R-N248D-E271F,S024R-S078D-G118S-G159D-Q245R-H249R,T022W-N043R-R045T-S101A-P210I-T213A,S024G-N076D-S078N-S101N-H120Q-S128A-L217Q,G020R-S024R-G118R-P129E-G159D-H249R, G020R-S078R-G159D-S188D, PI29E-S188D-Q245R-H249R, S078R-P129E-Q245R, S078R-P129E-N248D,T022A-S101Q-S103A-V104I-P129S-A232V-Q245R, S188D-N248D,S078R-G118R-G159D, G020R-S078G-G118S-Q245R-N248D-H249R,S024R-S078R-G118R-G159D, G020R-P129E-G159D-N248D,G020R-S024R-G159D-N248D, G097A-S128A-L217Q, S024R-P129E-G159D-N248D,T022K-S101A-S103N-V104L-A232T-Q245R, S078G-G118S-S188D-Q245R-H249R,S099T-S101G-G102A-S103A, G020R-G118R-G159D-Q245R,S024G-S078N-S101N-L217Q, S024G-S078N-S101N-S128A-L217Q-S256R,S078R-G118R-G159D-N248D, S078G-G118S-Q245R-H249R, G118R-P129E-N248D,G020K-T022L-Q245R, A098Q-S099T-S101G-S103A, G118R-H249R,S024R-G118R-N248D-H249R, G020R-S024R-S078G-G118S-Q245R-N248D-H249R,A098F-S099A-S101G-V104I, G020R-T022W-S024R-N043R-R045T-S101A-G211Q,A098Q-S099G-S101G-V104L,G020K-N062E-G097A-S101Q-Q109G-N116L-S188D-L217Q,G020K-N062E-S078N-G097A-S101Q-Q109G-N116L,G020K-N062E-G097A-S101Q-Q109N-N116L-L217Q,N043R-S078N-G102A-S128L-L217E-S240R, S078N-G097S-S101Q,N043R-N062E-S078N-S101D-S128L-S240R, S128L-L217E-E271L,G020K-S024G-N062E-G097S-S101N-Q109N-N116L-S128A-L217Q,G020K-N062E-N116L-S128L-T213A, S078N-G097A-S101G-Q109G-S188D-L217Q,S101D-S128L-L217E-S240R,G020K-S024G-N062E-G097A-S101Q-Q109G-N116L-S128A,G020K-N062E-S078N-G097S-S101N-Q109N-N116L-S128A-S188D-L217Q,G020K-N062E-G097S-S101Q-Q109G-N116L-S128A-S188D-L217Q,G020K-S024G-N062E-S078N-G097S-S101N-Q109N-N116L-S128A,S024G-S078N-G097S-S101G-S128A-S188D-L217Q,S024G-S078N-G097A-S101N-Q109G-S128A-S188D-L217Q,G020K-N043R-G102A-S128L, G020K-S078N-S101D-G102A-L217E-S240R,G020K-N062E-G100S, S078N-S128L-L217E-E271L, S128Q-L217E-E271L,G020K-S024G-N062E-S078N-G097A-S101N-N116L-S128A-L217Q,G020K-S024G-N062E-S078N-G097S-S101G-N116L-S128A-L217Q,G020K-S078N-S101D-G102A-L217E, G020K-N062E-S128L,G020K-N062E-S078N-S101G-Q109N-N116L,G020K-S024G-N062E-S078N-G097S-S101Q-N116L-S128A-L217Q,S078N-S128L-L217E-S240R, S101D-S128Q-L217E-S240R-E271L,N043R-G102A-S240R, G020K-N062E-S078N-S101N-Q109N-N116L-M222S,G020K-N062E-S101N-Q109G-N116L-S128A-L217Q,G020K-S024G-N062E-S078N-G097A-S101G-N116L-L217Q,G020K-N062E-S101G-N116L-S188D,G020K-N062E-G097S-S101Q-Q109N-N116L-S128A-S188D,G020K-I035V-G100S-S128L-L217E, N043R-S101D-G102A-S240R,N062E-S078N-G100S, G020K-N043R-N062E-A215T, N062E-S240R,N043R-S128L-L217E-S240R, N062E-S101D-S240R, G020K-S101D-N116L-T213A,G020K-S101D-N116L-S128Q-P210S-T213A, G020K-S101D-T213A,G020K-S024G-N062E-S078N-G097S-S101Q-Q109N-N116L-S128A,G020K-N062E-S078N-G097S-S101Q-N116L-S128A-L217Q,S024G-G097S-S101N-Q109G-S188D-L217Q,G020K-S024G-N062E-S101N-Q109G-N116L-L217Q, L217E-S240R,S101D-L217E-S240R-E271L, N043R-N062E-S101D-S128L-S240R,G097A-S101N-S128A-S188D-L217Q,G020K-N062E-S078N-S101N-N116L-S128A-L217Q,G020K-N062E-S078N-G097S-S101G-Q109N-N116L-L217Q,G020K-S024G-N062E-G097S-S101N-Q109G-N116L-S128A-L217Q,G020K-S024G-N062E-G097S-S101G-Q109G-N116L-S128A-S188D-L217Q,G020K-N062E-S101N-Q109N-N116L-S128A-S188D,S078N-G097S-S101N-Q109N-S128A-S188D-L217Q-W241L,G020K-N043R-N062E-S078N-S101D, G020K-N062E-S101D-S240R,G020K-S101D-S128Q, G020K-N116L-S128Q-Y209H-T213A,G020K-N043R-S078N-G100S-L217E-N218D,S024G-S101N-Q109G-S128A-S188D-L217Q,G020K-S024G-N062E-S078N-G097S-S101N-Q109G-N116L-S128A-S188D,S078N-S101N-Q109G-S188D-L217Q,G020K-S024G-N062E-S078N-G097S-S101N-Q109G-N116L-S188D-L217Q,G020K-N062E-S078N-S101Q-Q109N-N116L-S128A-S188D-L217Q,G020K-N062E-S101G-Q109G-N116L,G020K-S024G-N062E-G097S-S101N-Q109G-N116L-S128A-S188D-M222S,S024G-G097A-S101G-S188D-L217Q, S024G-S078N-G097A-S101G-S128A-S188D,G097S-S101N-Q109G-S128A-S188D, G020K-S078N-S101D-I246T,N043R-S128L-L217E, G020K-S101D-L217E-S240R, S128L-L217E-T224A-E271L,G020K-S078N-S101D-S128Q-L217E-E271L, N062E-S078N-S101D-L217E-S240R,G020K-S101D-L217E-E271L, G020K-S078N-S128Q-L217E-S240R,G020K-N062E-S101D-N116L-T213A, G100S-S101D,S078N-G097S-S101G-S128A-L217Q,G020K-N062E-G097S-S101N-N116L-S128A-L217Q,G020K-N062E-G097S-S101N-N116L-L217Q, S101N-Q109G-S188D,G020K-S024G-N062E-G097S-S101Q-Q109N-N116L-S128A-L217Q,G020K-S024G-N062E-G097S-S101N-N116L,G020K-S024G-N062E-S101N-N116L-S128A,G020K-S024G-N062E-S078N-S101Q-Q109N-N116L-S128A,G020K-N062E-S078N-S101N-Q109G-N116L-S188D,G020K-N062E-S078N-S101N-Q109G-N116L-S128A,G020K-S024G-N062E-S078N-G097S-S101N-Q109G-N116L-S128A-M222S,G020K-S024G-N062E-S078N-G097S-S101G-N116L-S128A,S024G-S078N-G097S-S101Q-Q109G-S128A-S188D-L217Q,S024G-S078N-S101G-Q109N-S188D-L217Q,S024G-S078N-G097A-S101G-Q109G-S188D,S078N-S101D-S128Q-L217E-S240R-E271L, G020K-S128Q-L217E,S128Q-L217E-S240R-E271L, G020K-S078N-S101D-L217E,G020K-S078N-S101D-L217E-E271L, G020K-N116L-S128Q-T213A,G020K-N116L-N123G, S101G-S188D-L217Q,G020K-S024G-N062E-S078N-S101G-N116L-L217Q,S078N-G097A-S101G-Q109N-S128A-S188D, G020K-S078N-S128L-L217E-E271L,G020K-N062E-S101Q-Q109G-N116L, G020K-S101D-L217E,S024G-G097A-S101N-Q109N-S128A, G020K-S078N-S101D-S128L-L217E-S240R,0097A-S101G-L217Q, S024G-S101Q-Q109G-S128A-L217Q,G020K-S024G-N062E-T071A-S078N-G097S-S101G-N116L-L217Q,G020K-S024G-N062E-S078N-S101G-N116L-S128A,G020K-S024G-N062E-S078N-G097A-S101N-N116L,G020K-N062E-G097A-S101N-N116L,G020K-S024G-N062E-G097A-S101N-Q109G-N116L-S128A,G020K-S024G-N062E-S078N-G097S-S101G-Q109N-N116L,G020K-N062E-S078N-G097S-S101Q-Q109G-N116L-S128A-S188D-L217Q,G020K-N062E-S101N-Q109G-N116L-L217Q,G020K-S024G-N062E-G097A-S101N-Q109G-N116L-S188D-L217Q,G020K-S024G-N062E-S101G-N116L, S078N-G097S-S101N-Q109G-G159D-L217Q,S024G-G097A-S101N-Q109G-S188D, S078N-S101G-S128A-L217Q,S078N-G097A-S101N-S128A-S188D-L217Q,S024G-G097S-S101G-S128A-S188D-L217Q,S024G-S078N-G097S-S101Q-Q109G-S128A-S188D-L217Q-V227A,G020K-N062E-S078N, S078N-S128L-L217E-S240R-E271L,G020K-N062E-I072N-S101D-N116L-S128Q,G020K-T057P-N062E-N116L-S128Q-T213A, G020K-N043R-S078N-S101D-S240R,S078N-G097S-S101N-Q109N-S128A-S188D-L217Q,S024G-S101N-Q109N-S128A-S188D,G020K-S024G-N062E-G097S-S101Q-N116L-S128A,S024G-S078N-G097S-S101N-Q109N-S128A-S188D, N043R-G100S,G020K-N062E-S078N-S101Q-Q109G-N116L-S128A,S078N-S101Q-Q109N-S188D-L217Q, G020K-N116L-S128L-T213A,G020K-S078N-S101D-L217E-S240R, G020K-N062E-S101N-Q109N-N116L-M222S,G020K-S024G-N062E-G097A-S101Q-Q109N-N116L-S128A,S078N-G097S-S101G-S128A-S188D, G097S-S101G-Q109N-S128A-M222S,S024G-S078N-G097A-S101Q-S128A-M222S, G097A-S101G-Q109N-L217Q,S024G-S078N-S101G-S128A-S188D-L217Q,S024G-G097A-S101N-Q109G-S128A-S188D, G097S-S101N-Q109G-S128A,S078N-G097A-S101Q-L217Q, G020K-N043R-S078N-S101D,G020K-N043R-E054D-N062E-S078N-S240R, G020K-S101D-S240R,S128Q-L217E-S240R, G020K-S128Q-L217E-V244A-E271L, G020K-L217E-E271L,G020K-S078N-S128L-L217E, S078N-S101D-S128Q-L217E-E271L,G020K-N062E-L217E-E271L, G020K-N062E-S101D-N116L-S128Q-T213A,G020K-N062E-S078N-N116L-S128I, G020K-N062E-S078N-S128Q-T213A,S024G-G097A-S101G-Q109G-S128A, S024G-G097S-S101N-Q109N-S128A-L217Q,G020K-S024G-N062E-S078N-S101N-N116L-S128A-S188D-L217Q, G020K-S101D,G020K-N062E-S078N-G097S-S101Q-N116L-S128A,G020K-N062E-G097S-S101G-N116L-S188D, N043R-S078N-G100S-S128L-S240R,G097S-S101N-Q109N-S188D, G097S-S101Q-Q109N-S128A-L217Q,G020K-N062E-S101N-Q109G-N116L-S128A-S188D-M222S,G020K-S024G-N062E-S078N-G097S-S101Q-Q109G-N116L, G020K-N062E-M222S,G097S-S101Q-Q109G-S188D, G020K-S078N-L217E-S240R,G020K-N062E-S078N-G097A-S101N-Q109G-N116L-S188D,G020K-S024G-N062E-G097S-S101Q-Q109G-N116L-S188D,G020K-N062E-S078N-G097A-S101Q-N116L-S128A-M222S,G020K-N062E-S078N-S101N-N116L-S188D,G020K-S024G-N062E-S078N-S101G-N116L,G020K-N062E-S078N-S101N-N116L-S128A-S188D,G020K-S024G-N062E-G097S-S101N-Q109G-N116L-S128A-S188D-L217Q,G020K-N062E-G097S-S101N-Q109N-N116L,G020K-N062E-S101G-N116L-S188D-L217Q, G097A-S101N-S128A,S101G-Q109N-S128A-M222S, S024G-G097S-S101Q-Q109G-S188D-L217Q,G097S-S101Q-Q109N-S128A-S188D, S024G-S078N-G097A-S101Q-S188D-L217Q,S024G-S078N-S101G-Q109G-S128A-L217Q, G020K-N062E-S128L-S240R,N062E-S078N-S128L-S240R, N043R-G102A-S128L-L217E-S240R,N062E-S078N-L217E-S240R-E271L, S078N-S128Q-L217E-N269S,G020K-N062E-N123G-T213A,G020K-N062E-S078N-S099G-N116L-S128Q-T213A-L217N,G020K-N062E-S078N-S128I-L217N, G097A-S101Q-S128A-S188D-L217Q,G020K-N062E-S078N-G097S-S101N-Q109G-N116L,G020K-S024G-N062E-S078N-G097S-S101N-Q109G-N116L-S188D,G020K-N043R-S128L-S240R, S024G-S078N-G097A-S101N-Q109N-S128A,G020K-N062E-G097S-S101Q-N116L-L217Q, G020K-N062E-S078N-S128I,G020K-S024G-N062E-L090V-G097A-S101Q-N116L-M222S,G020K-N062E-S078N-S101Q-Q109G-N116L-S188D-L217Q,G020K-S024G-N062E-G097A-S101G-N116L-S128A-S188D-L217Q,G020K-N062E-G097S-S101N-Q109N-N116L-S188D-L217Q,G020K-S024G-A048V-N062E-S078N-G097A-S101N-N116L-S128A-L217Q,G020K-N062E-S078N-S101Q-Q109G-N116L,G020K-S024G-N062E-G097S-S101N-Q109N-N116L-S128A,G020K-N062E-S078N-G097A-S101Q-Q109G-N116L-S188D,G020K-N062E-S078N-G097A-S101N-Q109N-N116L-N218D,S024G-S101N-Q109G-S128A-S188D-M222S,S024G-G097A-S101Q-Q109N-S188D-L217Q,S024G-S078N-G097S-S101Q-Q109N-S128A-S188D, S101N-Q109G-M222S,S024G-I072V-G097S-S101N-S128A-L217Q, S101G-S128A,N043R-S078N-G102A-A215G, G020K-S101D-S128L-L217E-S240R,S078N-S101D-S128Q-L217E-S240R, G020K-N116L-S128L-M222S,G020K-N062E-S078N-N116L-T213A-M222S, G020K-N062E-S078N-N116L-S128Q,G020K-N062E-T213A, or G020K-N062E-S078N-N116L-N123G-M222S (List 15), andwherein amino acid positions of the subtilisin variant are numberedaccording to the numbering of corresponding amino acid positions in theamino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ shownin SEQ ID NO:1.

The present invention provides an isolated subtilisin variant, whereinsaid subtilisin variant is a mature form having proteolytic activity andcomprises an amino acid sequence comprising a combination of amino acidsubstitutions at positions selected fromT022A-S024G-N076D-G097S-S101G-S103A-V104I-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,G020K-T022L-S078N-G118R-S128D-T213A,S024R-S078G-G118S-P129E-S188D-Q245R-H249R,T022A-N076D-S101G-S103A-V104I-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-S101G-S103A-V104I-S128A-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-S024G-G097S-S101Q-S103A-V104I-Q109G-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-N076D-S101N-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,T033S-G118R-S128I-A158E, T022A-S103A-L111V-L148I,T022A-N076D-A085T-S101G-S103A-V104I-P129E-S188D-A232V-Q245R-N248D-E271F,T022A-S024R-N076D-S101G-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-E271F,G020K-T022L-S078N-S128D-T213A-L217E, T022A-T033S-P129E-S166D,T022A-S024G-N076D-G097A-S101Q-S103A-V104I-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-G097A-S101N-S103A-V104I-Q109G-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,G020K-S078N-S166D-T213A,T022A-S101Q-S103A-V104I-L124V-P129S-G159D-S188D-A232V-Q245R-N248D-E271F,T022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245T-N248D,A158E-S166D-S188D,L021S-T022A-S024G-N076D-S101Q-S103A-V104I-S128A-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-G097A-S101N-S103A-V104I-L124V-S128A-P129E-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-S101G-S103A-V104I-G159D-S188D-A232V-N238Y-Q245R-N248D-A270V-E271F,T022A-T033S-N062E-N116L-S188D,T022A-S024G-S049N-N076D-S101G-S103A-V104I-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-G097S-S101Q-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,G097P-A098Q-S099A-S103G-V104I, G020K-S024F-G118R-S128D-S166D,T022A-S024G-S101G-S103A-V104I-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S024R-T033S-G118R-S166D,T022A-S024R-N076D-S101G-S103A-V104I-G159D-S188D-A232V-N238Y-Q245R-N248D-A270V-E271F,N062E-S078D-G118S-P129E-N248D-H249R,T022A-S024G-V026I-S101N-S103A-V104I-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,G097P-A098Q-S099A-S101A, S078G-G118D-P129E-S188D-Q245R-H249R,T022A-G097A-S101N-S103A-V104I-L126I-P129E-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,G020R-S024R-N062E-S078G-G118D-P129E-G159D-S188D-Q236R-Q245R-N248D,T022A-N076D-S078N-S101N-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-N076D-S101G-S103A-V104I-P129E-G159D-S188D-A232V-N238Y-Q245R-N248D-E271F-A272V,T022A-N076D-N077D-S078N-G097S-S101N-S103A-V104I-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-N043R-N076D-S101G-S103A-V104I-P129E-A158E-S188D-A232V-Q245R-N248D-A270V-E271F,S099G-S101A-G102A-S103A,T022A-S024G-G097A-S101N-S103A-V104I-Q109G-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,G020R-S024R-S078D-G118S-P129E-Q245R, S056C, S024R-T033S-N062E-N116L,T022A-S024G-N076D-S101G-S103A-V104I-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T033S-S101G-S103A-V104I-N116L-S128N-Y209A-T213A-A232V,A098Q-S099A-G100S-S101G-V104I,T022A-S078N-S101N-S103A-V104I-Q109G-S128A-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-S024R-N076D-S101G-S103A-V104I-G159D-S188D-Q245R-N248D-A270V-E271F-A272V,T022A-N076D-G097S-S101Q-S103A-V104I-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S024G-S078N-S101N-S103A-V104I-Q109N-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-L090I-S101G-S103A-V104I-S128A-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,G020R-N062E-S078G-G118D-P129E-G159D-S188D-N248D-H249R,A016S-T033S-S101G-S103A-V104I-N116L-S128N-Y209V-T213A-A232V,G020R-S078G-G118D-N248D-H249R,T022A-N062Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,S024R-N062E-N116L-A158E-S166D-S188D,T022A-S024G-N076D-S101Q-S103A-V104I-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-S024R-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-A270V-E271F,T022A-N062Q-S101G-S103A-V104I-P129E-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-N076D-S101N-S103A-V104I-Q109G-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-S101G-S103A-V104I-P129E-S188D-A232V-Q245R-N248D-E271F,T022A-S024G-N076D-S078N-G097A-S101N-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S024R-N076D-S101G-S103A-V104I-I107V-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S024R-N116L-G118R,T022A-S024R-T033S-N062E-N116L-G118R-S128I-A158E-S188D,T022A-N076D-G097S-S101N-S103A-V104I-Q109G-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S024G-N076D-G097S-S101Q-S103A-V104I-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,S024R-T033S-N062E-N116L-S128I-S188D,T022A-S024G-N076D-G097S-S101N-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,S240V, N062E-G118R,T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245T-N248E,S024R-N062E-S078G-G118D-P129E-S188D-Q245R-N248D, A016S-S103A-V104I,A088P,T022A-S101N-S103A-V104I-Q109N-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-S101N-S103A-V104I-L126I-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,G097P-A098F-V104L,T022A-G097S-S101N-S103A-V104I-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-G097A-S101N-S103A-V104I-Q109N-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,G020K-T022L-S128D,T022A-S024G-S101G-S103A-V104I-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,G020R-S024R-N062E-S078D-G118D-P129E-G159D-S188D-N248D-H249R,S024F-S078N-G118R-L217E,T022A-S024G-S078N-G097S-S101Q-S103A-V104I-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S024R-S101G-S103A-V104I-A158E-G159E-S188D-A232V-Q245R-N248D-E271F-A272V,S024R-N062E-N116L-G118R-S128I-S188D, G118R-S128D-S166D-T213A,T022A-G097A-S101G-S103A-V104I-P129E-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-T033S-S101G-S103A-V104I-N116L-S128L-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S078N-S101Q-S103A-V104I-Q109G-S128A-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-G097S-S101N-S103A-V104I-Q109G-S128A-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-S024G-G097S-S101N-S103A-V104I-Q109N-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-N076D-G097A-S101N-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S101G-S103A-V104I-L126I-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,G020R-S078G-G118S-S188D-H249R,T022A-S024G-N076D-S101Q-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S101A-S103A,T022A-N076D-S101G-S103A-V104I-P129E-G159E-S188D-A232V-Q245R-N248D,S024R-S078D-G118D-S188D-N248D-H249R, S099A-V104I,T022A-G097S-S101Q-S103A-V104I-Q109N-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,S024R-S078G-G118D-G159D-N248D,T022A-S024G-S078N-S101Q-S103A-V104I-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-N076D-S101G-S103A-V104I-P129E-G159E-S188D-A232V-Q245R-N248D-E271F,T022A-G097A-S101Q-S103A-V104I-L124V-S128A-P129E-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-G097A-S101Q-S103A-V104I-L124V-G159D-S188D-A232V-Q245R-N248D-E271F,S078G-G118D-P129E, S212W,T022A-S101G-S103A-V104I-I107V-P129E-G159D-S188D-A232V-N238Y-Q245R-N248D-A270V-E271F-A272V,T022A-S078N-S101Q-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-G097A-S101G-S103A-V104I-S128A-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,S101G-V104L,T022A-S101G-S103A-V104I-I107V-P129E-G159D-S188D-A232V-N238Y-Q245R-N248D-E271F,T022A-S024R-S101G-S103A-V104I-P129E-G159D-S188D-A232V-Q245R-N248D-E271F-A272V,T022A-S024G-G097A-S101Q-S103A-V104I-Q109N-S128A-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-S024G-N076D-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S024R-N076D-S101G-S103A-V104I-P129E-A158E-S188D-A232V-Q245R-N248D-E271F-A272V,T022A-T033S-S101G-S103A-V104I-N116L-N117S-Y209A-G211Q-T213A-A232V,S101G-S103A-V104I-N116L-G118V-Y209A-G211Q-A232V,T022A-N062E-S101N-S103A-V104I-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S024G-G097S-S101N-S103A-V104I-S128A-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,S024F-T213A,T022A-S024G-N076D-S101N-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-N076D-S101Q-S103A-V104I-Q109G-S128A-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-S078N-G097A-S099G-S101G-S103A-V104I-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-N076D-S101G-S103A-V104I-G159D-S188D-L217Q-N218S-A232V-Q245R-N248D-E271F,T022A-S024G-N076D-S101N-S103A-V104I-Q109G-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-S024G-S101N-S103A-V104I-Q109N-G159D-S188D-A232V-Q245R-N248D-E271F,G020R-S024R-S078D-G118D-Q245R,T022A-S078N-I079V-S101N-S103A-V104I-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-S024R-N116L-G118R-S128I-A158E-S166D, S078G-G118D-S188D,G097P-A098Q-S099G-G102A, S099A-S103G, S078D-G118D-P129E-Q245R-N248D,T022A-S024G-S078N-S101Q-S103A-V104I-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-S078N-S101N-S103A-V104I-Q109G-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-N076D-S078N-G097S-S101Q-S103A-V104I-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-S024R-N116L-S128I-A158E-S166D-S188D,A016S-T022A-S101A-S103G-V104I,T022A-G097S-S101N-S103A-V104I-S128A-G159D-R186H-S188D-A232V-Q245R-N248D-E271F,T022A-G097A-S101Q-S103A-V104I-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-S101N-S103A-V104I-L126I-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S101Q-S103A-V104I-L126I-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-N076D-G097S-S101N-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-N043R-N076D-S101G-S103A-V104I-P129E-S188D-A232V-Q245R-N248D-E271F-A272V,T022A-S024R-T033S-N062E-N116L-S128I-S188D,T022A-S024G-S078N-G097S-S101G-S103A-V104I-S128A-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022L-S078N-G118R-S128D-T213A,T022A-N076D-G097A-A098T-S101Q-S103A-V104I-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-S024G-S078N-G097A-S101Q-S103A-V104I-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,S103A-V104L, S024R-S078G-G118D-P129E-G159D-S188D-N248D,T022A-S024R-N043R-S101G-S103A-V104I-S188D-A232V-Q245R-N248D-E271F,T022A-S024R-G061W-N076D-S101G-S103A-V104I-P129E-S188D-A232V-Q245R-N248D-E271F,G020R-N062E-S078D-G118S-S188D-N248D,T022A-S101G-S103A-V104I-A158E-G159E-S188D-A232V-Q245R-N248D-E271F,G100S-V104I,T022A-S024G-N076D-S078N-G097S-S101N-S103A-V104I-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,G097P-A098F-S103A,T022A-G097S-S101Q-S103A-V104I-Q109G-G159D-S188D-A232V-Q245R-N248D-E271F,S024F-S078N-G118R-S128D-L217E,T022A-S101Q-S103A-V104I-Q109G-S128A-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,S024R-T033S-N116L-S128I-A158E-S166D, T022L-S024F-S128D,T022A-S101G-S103A-V104L,T022A-N043R-S101G-S103A-V104I-A158E-G159E-S188D-A232V-Q245R-N248D-E271F-A272V,T022A-N076D-S101G-S103A-V104I-I107V-G159D-S188D-A232V-Q245R-N248D-E271F,T022L-S024F-S078N, T022A-N062E-A158E-S188D, G020K-T022L-S166D-T213A,T022A-S024G-T071A-S078N-S101N-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,A016S-T022A-S101G-S103A-V104I-L148I,S078D-G118S-S188D-Q245R-N248D-H249R, G020K-T022L-S128D-S166D,T022A-S024G-N076D-G097S-S101N-S103A-V104I-Q109N-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-N062E-S101G-S103A-V104I-P129E-S188D-A232V-Q245R-N248D-E271F,G020R-N062E-S078G-G118D-P129E-Q245R-H249R,G020R-S078G-G118D-P129E-S188D-N248D,G020K-T022L-S024F-S078N-G118R-S128D-S166D,T022A-N076D-S101G-S103A-V104I-P129E-S188D-A232V-Q245R-N248D-E271F,T022A-S101Q-S103A-V104I-L126I-P129E-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T255G,T022A-G097A-S101N-S103A-V104I-L126I-S128A-P129E-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S024R-N076D-S101G-S103A-V104I-P129E-G159D-S188D-A232V-N238Y-Q245R-N248D-E271F,S024R-T033S-N062E-G118R-S166D-S188D,T022A-G097A-S101Q-S103A-V104I-S128A-P129S-G159D-S188D-A232V-Q245R-N248D-E271F,S024R-N062E-S078D-G118S-P129E-G159D-S188D-Q245R-H249R,T022A-S024G-S101Q-S103A-V104I-Q109N-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,A098Q-G102A,T022A-S024R-S101G-S103A-V104I-I107V-P129E-G159D-S188D-A232V-N238Y-Q245R-N248D-A270V-E271F,T022A-S024G-S078N-G097A-S101N-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S024G-S078N-S101N-S103A-V104I-Q109G-G159D-S188D-A232V-Q245R-N248D-E271F,S024R-N116L,T022A-S101G-S103A-V104I-L126I-P129E-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,G020K-S078N-S128D,T022A-S024G-S101N-S103A-V104I-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S024G-S078N-S101Q-S103A-V104I-S128A-G159D-S188D-T213A-L217Q-A232V-Q245R-N248D-E271F,T022A-S024G-S101N-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S101N-S103A-V104I-L126I-P129E-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-S024G-N076D-G097A-S101N-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S024G-N076D-S078N-G097A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,S024R-T033S-N062E-N116L-S166D,T022A-S024G-N076D-S101N-S103A-V104I-Q109G-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,A016S-S024F-S101G-S103A-V104I-N116L-Y209V-A232V,G020K-S024F-S078N-S128D-S166D, G025A,T022A-S024R-N076D-S101G-S103A-V104I-G159D-S188D-A232V-N238Y-Q245R-N248D-E271F,G097P-S099T-V104I,T022A-N076D-S078N-G097A-S101N-S103A-V104I-Q109G-G159D-S188D-A232V-Q245R-N248D-E271F,G020R-S024R-S078G-G118S-G159D-Q245R-N248D-H249R,T022A-S024G-S078N-S101Q-S103A-V104I-Q109G-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-S024G-N076D-G097A-S101Q-S103A-V104I-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-N076D-S078N-G097S-S101N-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S078N-G097S-S101G-S103A-V104I-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S078N-G097S-S101Q-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,D041K,T022A-S024G-G097A-S101G-S103A-V104I-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S024G-G097S-S101N-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,A016S-T022A-S101A-V104I-L111V-L148I, A016S-T022A-S101A-V104L-L111V,T022A-S024G-G097S-S101Q-S103A-V104I-Q109N-G159D-S188D-A232V-Q245R-N248D-E271F,S078G-G118S-S188D-N248D, 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D041L,S024R-N062E-S128I-A158E-S188D, N062E-S078D-G118D-N248D-H249R,G020K-S024F-G118R-T213A, S101G-S103G-V104I-S128N,S078N-G118R-S128D-T213A-L217E,T022A-N076D-S101G-S103A-V104I-Q109N-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-N076D-G097S-S103A-V104I-Q109N-G159D-S188D-A232V-Q245R-N248D-E271F,S078G-G118S-G159D-N248D, T033S-A158E,T022A-S101G-S103A-V104I-I107V-G159D-S188D-A232V-Q245R-N248D-E271F-A272V,T022A-S101G-S103A-V104I-A158E-G159E-S188D-A232V-Q245R-N248D-E271F-A272V,T022A-S024R-T033S-S128I-S166D-S188D,T022A-N076D-S101G-S103A-V104I-A158E-G159E-S188D-A232V-Q245R-N248D-E271F,T022A-S024G-S078N-G097S-S101Q-S103A-V104I-Q109G-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S024R-N076S-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F-A272V,S024R-G118R-S128I-S166D, T033S-N116L-G118R-S128I-S188D,T022A-S101G-S103A-V104I-G159D-S188D-A232V-N238Y-Q245R-N248D-E271F-A272V,T022A-S024R-T033S-N116L-G118R-V139I-S188D,T022A-S024R-N076D-S101G-S103A-V104I-P129E-A158E-G159E-S188D-A232V-Q245R-N248D-E271F,N062E-S078G-G118S-P129E-G159D-S188D-Q245R-N248D,T022A-S024G-S078N-G097A-S101N-S103A-V104I-Q109G-S128A-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-N062Q-S101Q-S103A-V104I-S128A-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-S024G-G097A-S101G-S103A-V104I-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,A016S-T022A-S024F-S101G-S103A-V104I-N116L-S128N-Y209A-T213A-A232V,T022A-G097A-S101Q-S103A-V104I-L124V-S128A-P129E-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,S024R-N062E-N116L-G118R-S128I-S166D-S188D,T022A-S024R-T033S-N116L-G118R-S128I-A158E-S166D, A108V-S128D-T213A,T022A-N043R-N076D-S101G-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-E271F-A272V,T022A-S101G-S103A-V104I-I107V-G159D-S188D-A232V-Q245R-N248D-A270V-E271F,T022A-N076D-S101G-S103A-V104I-A158E-G159E-S188D-A232V-Q245R-N248D-E271F-A272V,T033S-S101G-S103A-V104I-N116L-S128N-Y209A-A232V,S024F-T033S-S101G-S103A-V104I-I107V-N116A-Y209V-T213A-A232V,T022A-S024G-G097S-S101N-S103A-V104I-Q109N-G159D-S188D-A232V-Q245R-N248D-E271F,S024R-T033S-N116L-S128I-S166D-S188D,T022A-S024R-N076D-S101G-S103A-V104I-G159D-S188D-A232V-N238Y-Q245R-N248D-E271F-A272V,T022A-G097A-S101Q-S103A-V104I-L126I-S128A-P129E-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S078N-G097A-S101Q-S103A-V104I-Q109G-G159D-S188D-A232V-Q245R-N248D-E271F,A098Q-S099G-G102A,T022A-S024G-G097A-S101G-S103A-V104I-P129E-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-S024G-S101N-S103A-V104I-Q109G-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-G097S-S101Q-S103A-V104I-S128A-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-S024R-S101G-S103A-V104I-I107V-G159D-S188D-A232V-Q245R-N248D-E271F,T022L-G118R-S166D, V011M-S024R-T033S-S128I-S166D,G020K-S078N-S128D-L217E, V004M, N062E-N116L-G118R-S128I-S166D,T022A-G097A-S101N-S103A-V104I-Q109G-S128A-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,G020R-S024R-N062E-S078G-G118S-P129E-Q245R-N248D-H249R,T022A-S101G-S103A-V104I-P129E-G159D-S188D-A232V-N238Y-Q245R-N248D-E271F,T022A-S101N-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-T033S-N116L-S166D-S188D,T022A-S078N-G097S-S101Q-S103A-V104I-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-N076D-S078N-G097S-S101N-S103A-V104I-Q109G-S128A-G159D-S188D-A232V-Q245R-N248D-E271F,G118R-T213A,T022A-S101N-S103A-V104I-L126I-G159D-S188D-A232V-Q245R-N248D-E271F,S024R-N116L-S188D,T022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245V-N248E,T022A-S024G-N076D-G097S-S101Q-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-T033S-S101G-S103A-V104I-N116L-G159D-S188D-A232V-Q245R-N248D-E271F,G097P-S099T-S101A-V104L,T022A-N076D-S101G-S103A-V104I-P129E-G159D-S188D-A232V-N238Y-Q245R-N248D-A270V-E271F,T022A-S024G-G097A-S101N-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,T022A-N043R-N076D-S101G-S103A-V104I-S188D-A232V-Q245R-N248D-E271F-A272V,G020R-S024R-S078G-G118D-G159D-Q245R-N248D-H249R,T022A-S101G-S103A-V104I-G159D-S188D-A232V-N238Y-Q245R-N248D-E271F,L021F-T022A-S024G-S078N-S101Q-S103A-V104I-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,N062E-S078G-G118D-G159D-S188D-Q245R-N248D, A098Q-S101A-S103A-V104L,T022A-S024G-S078N-S101Q-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,N062E-G118R-A158E-S166D-S188D,T022A-S024G-S078N-S101Q-S103A-V104I-Q109N-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,N116L-S128I, 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T022A-S166D-S188D,S078N-G118R-S128D-S166D-T213A,T022A-S024G-S078N-G097A-S101N-S103A-V104I-Q109N-S128A-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F,T022A-N043R-S101G-S103A-V104I-G159E-S188D-A232V-Q245R-N248D-E271F,G020R-S024R-N062E-S078D-G118D-G159D-S188D-N248D,G097P-S099T-S101A-S103G-V104I,T022A-S024R-N043R-N076D-S101G-S103A-V104I-P129E-S188D-A232V-Q245R-N248D-E271F,A016S-T022A-S101A-S103G-V104L-S128N,G020R-N062E-S078G-G118S-G159D-S188D-Q245R-H249R, G020R-S078G-G118D,G020R-S078G-G118S, Q012Y-N018G,T022A-S024R-N043R-N076D-S101G-S103A-V104I-P129E-A158E-S188D-A232V-Q245R-N248D-E271F,T033S-N062E-G118R-A158E-S166D,T022A-N076D-A088V-S101Q-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F,S024R-N062E-N116L-S128I-S166D-S188D, T022A-T033S-N116L-A158E,T022A-S024R-N043R-N076D-S101G-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-E271F-A272V,T033S-N062E-N116L-A158E, 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S024G-S078N-S101G-Q109G-M222S,S078N-G097A-S101G-Q109G-S128A, S078N-G097S-S101N-Q109N-S128A,S078N-G097A-S101N-Q109N-S188D, S024G-S101N-S128A-S188D-M222S,S101Q-Q109N-S188D-M222S, S101D-G102A-L217E,G020K-N062E-S078N-S101D-L217E, G020K-S078N-S101D-I107T-S128L-S240R,G020K-S078N-G100S-L217E-N218D, G020K-N043R-S101D,N043R-G100S-S128L-L217E, G020K-S078N-S101D-S128L-L217E-E271L,G020K-S101D-S128L-E271L, G020K-S101D-N116L-S128L-T213A,G020K-N062E-S078N-T213A-L217N, G020K-N062E-N116L-L217N,G020K-N062E-S128I-T213A, N062E-S101D-S128L-S240R, G097S-S101N-S128A,G020K-N062E-S078N-N116L-T213A-L217N, G020K-S101D-M222S,S024G-S101G-Q109G, G097A-S101Q-Q109G, G097S-S101Q-Q109N-S188D,G020K-N062E-G097S-S101N-N116L-S128A-S188D-L217Q,S078N-S101Q-S128A-M222S, S078N-G097A-S101Q-Q109N,S024G-S078N-G097A-S101Q-S128A-S188D, S078N-S101D-G102A,G020K-N062E-S078N-G097S-S101Q-Q109G-N116L-S128A-S188D-M222S,G020K-S024G-N062E-S078N-G097S-S101N-Q109N-N116L-S188D-L217Q-M222S,G020K-N062E-G097S-S101Q-N116L-S128A,G020K-S024G-N062E-G097S-S101Q-N116L-S128A-S188D-L217Q,S024G-S101N-Q109N-S128A, S024G-G097S-S101Q-S128A-S188D,S024G-S101Q-L217Q, G097S-S101G-S128A, S024G-S078N-S101Q-S128A-L217Q,S024G-S078N-G097S-S101N-S188D, S024G-G097A-S101N-S128A-M222S,G097A-S101Q-Q109N-S188D-M222S, S024G-S078N-G097S-S101Q,G020K-S101D-S128L-S212P-L217E-S240R,G020K-S101D-S128L-L217E-N218S-S240R, S101D-S128Q,G020K-N062E-S078N-E271L, S101D-E271L, S101D-S240R-E271L,G020K-S101D-E271L, G020K-N116L-S128L-T213A-M222S,G020K-N116L-S128Q-M222S, G020K-N062E-V084A-S128Q-M222S,G020K-N062E-S078N-N116L, S078N-S101N-Q109G-S128A-M222S,S024G-G097S-S101Q-Q109G, G020K-N062E-T213A-L217N,G097S-S101G-S128A-S188D, G020K-S240R, N062E-S078N, G020K-S128Q-M222S,S078N-G097S-S101G-Q1090, G020R-S024G-N043R-S078N-S101Q,G097S-S101N-Q109G-S128A-M222S, S024G-G097S-S101G-Q109N,S024G-S078N-G097A-S101G-Q109N,G020K-N062E-S078N-S101N-N116L-S128A-S188D-L217Q,G020K-N062E-S101Q-Q109G-N116L-S128A-M222S,G020K-S024G-N062E-S101Q-Q109N-N116L, S024G-S078N-S101Q-M222S,S024G-G097A-S101Q-Q109G-S128A, S101Q-Q109N-L217Q,S101Q-Q109G-S188D-L217Q, S024G-G097A-S101N-S188D-M222S,S024G-S078N-G097A-S101Q-Q109N, G097A-S101Q-S128A,G097A-S101Q-Q109G-S128A, S024G-G097S-S101Q-M222S,S078N-S101D-L217E-S240R, G020K-G100S-S128L,G020K-N043R-S078N-S128L-S240R, G020K-N043R-S101D-S240R,G020K-N043R-S078N-S128L, N062E-S128L, G020K-S078N-S101D-S128L,S078N-S101D, G020K-S101D-N116L-T213A-M222S, G020K-T213A-M222S,G020K-N116L-T213A, 0020K-N116L, G020K-N062E-S128Q-L217N,S078N-G097S-S101G-Q109N-S128A, S078N-S101Q-Q1090, N062E-S078N-S128L,G020K-N062E-N116L-S128Q-T213A-L217N, G020K-N116L-T213A-M222S,G020K-S024G-N062E-S101Q-Q109H-N116L-M222S,G020K-N116L-S128Q-T213A-M222S, S024G-G097S-S101N,G020K-P055T-N062E-N116L-L217N, S024G-S101Q-S128A-S188D-M222S,G020K-M222S, G020K-N062E-S078N-G097A-S101N-N116L-S188D-L217Q,G097S-S101G-Q109N-M222S,G020K-S024G-N062E-S078N-G097A-S101N-N116L-S128A-L217Q-M222S,G020K-S024G-N062E-G097S-S101N-Q109N-N116L-S128A-S188D-L217Q-M222S,G020K-N062E-S078N-S101Q-N116L-S128A, S024G-S078N-S10IN-Q109G-S188D-L217Q-M222S, S101N-Q109N-S128A-M222S,S101Q-Q109G-S128A-S188D, G097A-S101G-S188D-M222S,S078N-G097S-S101N-Q109G-S128A-S188D-L217Q-M222S,S024G-G097A-S101Q-M222S, S024G-G097S-S101N-Q109N-S128A-M222S,S024G-S078N-G097S-S101N-M222S, G020K-G102A-S240R, S078N-S128Q-E271L,G020K-N062E-S078N-S128Q, G020K-N062E-S078N-T213A-M222S,G020K-N062E-S078N-S128I-T213A-L217N, S078N-G097A-S101N,G020K-N062E-N116L-S128I-T213A-L217N,S024G-G097A-S101Q-S128A-S188D-M222S, S024G-S078N-G097A-S101N,S024G-S078N-G097S-S101G-M222S,G020K-N062E-S078N-N116L-S128Q-T213A-M222S, G020K-N062E-S128Q-M222S,G020K-N062E-S101Q-N116L, G020K-N062E-S101Q-Q109G-N116L-S188D-L217Q,G020K-S024G-N062E-G097A-S101N-N116L-S128A-S188D,G020K-S024G-N062E-S078N-S101Q-Q109G-N116L-S188D-M222S,S078N-G097S-S101N-Q109G-S128A-L217Q-M222S,S024G-S078N-S101N-Q109G-S128A-L217Q-M222S, G097A-S101Q-S128A-S188D,S024G-S078N-G097A-S101N-Q109N-S128A-S188D-L217Q-M222S,S024G-S078N-G097A-S101G-L217E-M222S, S078N-G097S-S101Q-Q109G-T274I,G097A-S101G-M222S, S101N-Q109N, G020K-L217E-S240R-E271L,S128Q-S240R-E271L, G020K-S101D-N116L-S128Q-M222S,G020K-P055L-N062E-N116L-S128I, G020K-N062E-S101G-N116L-S128A-M222S,G020K-S078N-S101D-S128L-S240R-E271L,G020K-N062E-S078N-G097S-S101Q-Q109N-N116L-L217Q-M222S,S078N-S101D-S240R-E271L, S024G-S078N-S101N-Q109G-L217Q-M222S,G097A-S101G-Q109G-L217Q-M222S,G020K-S024G-N062E-S078N-G097S-S101N-N116L-S128A-L217Q-M222S,G020K-S078N-S128L-E271L, G020K-N062E-S078N-N116L-L217N,G097A-S101G-Q109G-S188D-L217Q-M222S,S024G-S078N-G097S-S101N-Q109G-S128A,G020K-S024G-N062E-S078N-G097S-S101N-N116L-S188D-M222S,G020K-S024G-N062E-G097S-S101Q-N116L-S128A-M222S,G020K-N062E-S078N-G097A-S101G-Q109G-N116L-S128A-S188D-L217Q,G020K-N062E-S101G-N116L-S128A-L217Q-M222S,S024G-G097A-S101Q-S188D-M222S, S024G-S101G, T072V-S101Q-S128A,S078N-G097S-S101N-Q109N, S024G-G097A-S101N-Q109N-S188D-L217Q-M222S,S078N-S101Q-Q109N-S188D-L217Q-M222S, G020K-N043R-N062E-S128L-L217E,N043R-G102A-S128L-S240R, 0020K-N116L-N123G-T213A-M222S,G020K-S101D-S128Q-T213A-M222S, S024G-G097A-S101Q-Q109G, G020K-S078N,G020K-N062E-S101Q-N116L-S188D,G020K-N062E-S078N-G097S-S101N-Q109G-N116L-S188D-L217Q-M222S,G020K-S024G-N062E-G097S-S101G-N116L-S128A-L217Q-M222S,G020K-N062E-G097A-S101N-N116L-S128A-L217Q-M222S,S078N-G097S-S101G-Q109N-M222S, S024G-G097A-S101Q-Q109N,S024G-G097S-S101N-Q109N-S128A-S188D-L217Q-M222S,S078N-S101N-Q109N-S188D-L217Q-M222S, S078N-S101D-S128L-L217E-S240R,G020K-N062E-S101D-N116L-S128L, G097S-S101N-S188D-M222S,G020K-S078N-S128L-S240R-E271L, G020K-N062E-N116L-S128I-T213A,G020K-S024G-N062E-S078N-G097S-S101Q-N116L-S188D-M222S,G020K-N062E-S101Q-Q109G-N116L-L217Q-M222S,G020K-S024G-N062E-G097A-S101Q-N116L-L217Q-M222S,G020K-N062E-S078N-G097S-S101Q-N116L-S128A-S188D-M222S-A232T,G020K-S024G-N062E-S078N-G097S-S101Q-Q109N-N116L-S128A-L217Q-M222S,G020K-N062E-G097A-S101Q-N116L-S188D-M222S,S024G-S078N-G097S-S101N-Q109G-S128A-L217Q-M222S,S024G-G097A-S101Q-Q109N-S128A-S188D-L217Q-M222S, G020K-N043R,G020K-N043R-S078N-G102A-S128L, G097R-S101D,G020K-N062E-S078N-I107V-N123G-S128I,G020K-N062E-S078N-N116L-N123G-T213A, G020K-N062E-N116L-S128I-M222S,G020K-N043R-S240R, S078N-S101D-S128L, G020K-S078N-S128Q-E271L,S078N-S101G-S128A-S188D-L217Q-M222S,S078N-G097S-S101G-Q109N-S128A-L217Q-M222S, orG020K-N062E-N116L-S128Q-T213A-M222S (List 16), and wherein amino acidpositions of the subtilisin variant are numbered according to thenumbering of corresponding amino acid positions in the amino acidsequence of Bacillus amyloliquefaciens subtilisin BPN′ shown in SEQ IDNO:1.

The present invention further provides an isolated subtilisin variant,wherein said subtilisin variant is a mature form having proteolyticactivity and comprises an amino acid sequence comprising a combinationof amino acid substitutions at positions selected fromG020R-T022W-S024R-R045T-S101A-N204S-G211Q,T022W-S024R-S101A-P210I-T213A, G020R-S101A-G211Q,G020R-S024R-S101A-P210I-G211Q-T213A, G020R-S101A-P210I-T213A,T022W-S024R-N043R-S101A-P210I, G020R-S024R-N043R-R045T-S101A-G211Q,T022R-S101T-S103N-V104L-A232M-Q245R,G020R-T022A-S024R-R045T-S101A-T213A,T022R-S101G-S103N-V104I-A232M-Q245R,G020R-T022W-S024R-S101A-N116L-G211Q-T213A,G020R-T022W-N043R-S101A-N116L-G211Q, G020R-T022W-N043R-R045T-S101A,T022W-N043R-R045T-S101A-P210I-T213A,G020R-S024R-R045T-S101A-Q109R-P210I-G211Q-T213A,S024R-N043K-S101A-N204D-P210I, G020R-S024R-R045T-S101A-P210I,S024R-N043R-R045T-S101A-P210I-T213A, G020R-S024R-R045T-S101A-T213A,T022R-S101A-S103N-V104I-A232T-Q245R,G020R-T022W-S024R-R045T-S101A-N204S-P210I-G211Q,T022W-N043R-S101A-P210I-G211Q,G020R-S024R-N043K-R045T-S101A-N116L-P210I,T022A-S101G-S103A-V104I-I107V-L217Q-A232V-Q245R,S024R-N043R-R045T-S101A-G211Q-T213A,T022R-S101A-S103G-V104I-A232M-Q245R,T022R-S101G-S103N-V104L-A232V-Q245R,S024R-S078G-G118S-Q245R-N248D-H249R, S024R-N043R-S101A-P210I-T213A,T022K-S101N-S103A-V104I-A232T-Q245R, G020R-N043R-S101A-P210I-G211Q,T022R-S101T-S103A-V104I-A232M-Q245R,S024R-V026A-N043R-S101A-P210I-G211Q,T022A-S078N-S101G-S103A-V104I-S128A-L217Q-A232V-Q245R,T022K-S101T-S103G-V104I-A232L-Q245R,G020R-T022W-S024R-N043R-R045T-S101A-G211Q,G020R-S024R-S078G-G118D-Q245R,T022A-S101N-S103A-V104I-S128A-P129S-A232V-Q245R,T022A-G097A-S101G-S103A-V104I-L217Q-A232V-Q245R,G020R-T022W-N043R-S101A-G211Q-T213A,N018K-G020R-S024R-R045T-S101A-P210I-G211Q-T213A,G020R-T022W-S024R-N043R-R045T-S101A,T022R-S101N-S103A-V104L-A232T-Q245R, G020R-S024R-S101A-T213A,T022R-S101N-S103G-V104I-A232V-Q245R,G020R-T022W-S024R-S101A-G211Q-T213A,T022A-S024G-S101G-S103A-V104I-S128A-L217Q-A232V-Q245R,S024R-S078G-G118D-P129E-Q245R-H249R,T022R-S101T-S103A-V104L-A232L-Q245R, G020R-S024R-S101A-G211Q-T213A,T022A-S024G-S101G-S103A-V104I-L217Q-A232V-Q245R,G020R-T022W-N043R-V051A-S101A-P210I-G211Q, S024R-S078G-G118D-Q245R,N043R-S101A-P210I, T022Y-S101G-S103N-V104I-A232L-Q245R,G020R-T022W-N043R-S101A-T213A,G020R-S024R-S078D-G118S-S188D-Q245R-H249R,T022Y-S101N-S103N-V104I-A232T-Q245R,G020R-T022W-S024R-F050L-S101A-G211Q-T213A,N018S-T022W-S024R-N043R-S101A-G211Q,T022A-S101G-S103A-V104I-S128A-P129S-L217Q-A232V-Q245R,T022R-S101N-S103N-V104I-A232V-Q245R,T022A-S024G-S078N-G097S-S101Q-S103A-V104I-L217Q-A232V-Q245R,S101A-P210I-G211Q-T213A, G020R-N043R-R045T-S101A-P210I-G211Q-T213A,T022R-S101A-S103N-V104I-A232M-Q245S,G020R-T022W-N043R-R045T-S101A-G211Q,G020R-T022W-N043R-R045T-S101A-N116L-P210I,G020R-S024R-S078D-G118S-Q245R,G020R-T022W-S024R-N043R-S101A-P210I-G211Q-T213A,G020R-S024R-N043R-R045T-V051A-S101A-P210I-G211Q,T022A-S101G-S103A-V104I-P129S-L217Q-A232V-Q245R,G020R-S078G-G118D-Q245R-N248D-H249R,T022K-S101A-S103N-V104L-A232T-Q245R,T022A-N076D-S078N-G097S-S101G-S103A-V104I-L217Q-A232V-Q245R,S024R-N043R-R045T-S101A-P210I,T022A-S024G-G061R-S078N-S101G-S103A-V104I-S128A-A232V-Q245R,T022A-S078N-S101G-S103A-V104I-L217Q-A232V-Q245R,T022A-S024G-S078N-S101Q-S103A-V104I-S128A-L217Q-A232V-Q245R,T022Q-S101T-S103A-V104L-A232V-Q245R,G020R-T022W-S024R-R045T-S101A-G211Q, PO 14R,T022R-S101A-S103N-V104L-A232M-Q245R,T022K-S101G-S103N-V104I-A232V-Q245R, G020R-S024R-S078D-G118D-Q245R,T022A-G097S-S101N-S103A-V104I-L217Q-A232V-Q245R,G020R-S024R-S101A-P210I-T213A, T022K-S101N-S103N-V104I-A232V-Q245R,T022Y-S101T-S103A-V104I-A232L-Q245R,T022A-N076D-S078N-S101G-S103A-V104I-S128A-L217Q-A232V-Q245R,G020R-S024R-N043R-R045T-S101A-P210I-G211Q-T213A,G020R-R045T-S101A-P210I-T213A, G020R-S078D-G118S-Q245R-H249R,G020R-T022W-R045T-V051A-S101A, T022R-S101T-S103A-V104I-A232V-Q245R,N043R-S101A, G020R-S024R-N043R-R045T-S101A-G211Q-T213A,T022R-S101G-S103G-V104I-A232L-Q245R, G020R-S101A-P210I,T022K-S101T-S103N-V104I-A232V-Q245R, G020R-T022W-N043R-V051A-S101A,G020R-T022W-N043R-S101A, G020R-T022W-S024R-N043R-S101A-P210I,G020R-T022W-S024R-N043R-R045T-S101A-P210I-T213A,G020R-N043R-R045T-S101A-T213A,T022A-S024G-S078N-S101N-S103A-V104I-L217Q-A232V-Q245R,T022Y-S101N-S103N-V104L-A232T-Q245R, G020R-S101A-N116L-P210I-G211Q,T022R-S101A-S103N-V104L-A232L-Q245R, G020R-S024R-N043R-S101A,T022R-S101G-S103A-V104L-A232T-Q245R, G020R-S024R-S101A-G211Q,T022A-S101Q-S103A-V104I-P129S-A232V-Q245R,T022A-S024G-G097A-S101G-S103A-V104I-L217Q-A232V-Q245R,T022A-S024G-S078N-S101G-S103A-V104I-S128A-L217Q-A232V-Q245R,T022K-S101A-S103N-V104I-A232T-Q245R, G020R-T022W-S024R-R045T-S101A,T022Y-S101G-S103A-V104I-A232V-Q245R,S024R-N043R-R045T-S101A-N116L-P210I-G211Q-T213A,T022A-S024G-S078N-S101N-S103A-V104I-S128A-L217Q-A232V-Q245R,G020R-N043R-S101A-P210I,T022A-S024G-S078N-G097A-S101G-S103A-V104I-L217Q-A232V-Q245R,T022R-S101T-S103N-V104I-A232V-Q245R, P055R,T022A-S101N-S103N-V104I-A232V-Q245R,G020R-T022W-S024R-N043R-N076D-S101A-P210I-T213A,G020R-T022W-S101A-P210I-T213A, 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S024R-S078D-G118D-Q245R-H249R,G020R-S024R-N043R-R045T-S101A-Q109R-T213A,T022A-S078N-S101N-S103A-V104I-Q109N-A232V-Q245R,G020R-S024R-N043R-S101A-N204D-G211Q-T213A,T022A-G097A-S101Q-S103A-V104I-A232V-Q245R,G020R-S024R-S078G-G118S-N248D,G020R-S024R-S078G-G118S-G159D-Q245R-N248D,T022A-S024G-S078N-S101N-S103A-V104I-S128A-A232V-Q245R,S024R-S078G-G118D-G159D-Q245R-H249R,T022K-S101T-S103N-V104L-A232V-Q245R, S024R-S078G-G118S-Q245R,T022A-S101A-S103G-V104I-A232L-Q245R,T022A-G097A-S101Q-S103A-V104I-S128A-A232V-Q245R,T022A-S024G-S078N-G097S-S101G-S103A-V104I-A232V-Q245R,G020R-S078D-G118S-Q245R,T022A-S024R-N076D-S101G-S103A-V104I-A232V-N238Y-Q245R-A270V-A272V,S101A-P210I-T213A,T022A-S101Q-S103A-V104I-S128A-P129S-L217Q-A232V-Q245R, A114L,A016S-T022A-S101G-S103A-V104I-N116A-Y209V-G211Q-T213A-A232V,T022A-S101N-S103A-V104I-S128A-P129S-L217Q-A232V-Q245R,T022R-S101A-S103N-V104I-A232L-Q245S,T022Q-S101N-S103A-V104I-A232M-Q245W,T022W-S024R-S101A-N204S-G211Q-T213A,T022A-S101N-S103A-V104L-A232M-Q245R,T022A-S024G-S101Q-S103A-V104I-Q109N-S128A-A232V-Q245R,G020R-S078D-G118S-P129E-Q245R-H249R, T022Q-S101T-S03A-V104I-A232T-Q245S,S024R-N043R-R045T-S101A-P210I-G211Q-T213A,T022R-S101N-S103G-V104I-A232T-Q245R, S024R-S101A-P210I-G211Q-T213A,T022A-S024G-N076D-G097A-S101G-S103A-V104I-S128A-A232V-Q245R,G020R-S024R-S078G-G118S-S188D-N248D-H249R,T022A-G097S-S101Q-S103A-V104I-Q109G-S128A-A232V-Q245R,G020R-S024R-R045T-S101A-P210I-T213A,T022A-S078N-G097S-S101N-S103A-V104I-S128A-A232V-Q245R,T022A-S024G-S078N-G097A-S101Q-S103A-V104I-A232V-Q245R,T022R-S101G-S103N-V104I-A232T-Q245W,T022K-S101G-S103A-V104I-A232V-Q245R, T022W-S024R-S101A-G211Q-T213A,S024R-N043R-S101A-P210I-G211Q, T022Q-S101T-S103G-V104I-A232T-Q245R,T022A-S101G-S103A-V104I-A232V-Q245R-A272V,T022K-S101G-S103N-V104L-A232M-Q245R,T022A-S024G-G097A-S101Q-S103A-V104I-Q109N-A232V-Q245R,S078D-G118S-G159D-Q245R-H249R,G020R-T022W-S024R-A069T-S101A-P210I-T213A,T022A-S101N-S103A-V104I-S128A-L217Q-A232V-Q245R,G020R-T022W-S024R-N043R-R045T-S101A-P210I-G211Q,G020R-S024R-S078D-G118D-G159D-S188D-Q245R-H249R,T022A-S078N-S101N-S103A-V104I-S128A-L217Q-A232V-Q245R,G020R-S078G-G118D-G159D-Q245R-N248D-H249R, T022W-R045T-S101A-T213A,G020R-S024R-S078N-G097A-S101A-Q109N-N116L-L217Q,T022R-S024F-S101G-S103A-V104I-A232V-Q245R-E271H,G020R-S024R-S078N-G097S-S101Q-S128A-L217Q,T022R-S101G-S103A-V104I-A232V-Q245R-E271H,G020R-S024R-S101G-N116L-S128A, G020R-S024R-S101A-Q109N-N116L-S128A,T022R-S024F-S101G-S103A-V104I-A232V-Q245R-T260M,G020R-S024R-S078N-S101N-N116L-S128A,G020R-S024R-G097A-S101A-Q109G-N116L, G020R-S024R-S101N-S128A,G020R-S024R-S078N-S101G-S128A-L217Q,T022R-S024F-S101G-S103A-V104I-A232V-Q245R,T022R-S078N-G097S-S101Q-S103A-V104I-Q109N-N116L-A232V-Q245R,G020R-S024R-G097A-S101A-Q109N-N116L-S128A,G020R-S024R-G097S-S101N-Q109N-S128A,T022R-S101G-S103A-V104I-A232V-P239G-Q245R,G020R-S024R-G097S-S101A-S128A, G020R-S024R-S101G-Q109N-N116L-A215V,G020R-S024R-S078N-G097S-S101Q-Q109G-N116L-S128A,G020R-S024R-S078N-G097S-S101N-Q109N, G020R-S024R-S101G-S128A,T022R-S101G-S103A-V104I-A232V-Q245R-T260K-L267N,T022R-S101G-S103A-V104I-S128A-A232V-Q245R,T022R-S024G-G097S-S101N-S103A-V104I-A232V-Q245R,T022R-S024F-S101G-S103A-V104I-V121F-A232V-Q245R,G020R-S024R-G097A-S101Q-N116L-S128A-L217Q,T022R-G097A-S101N-S103A-V104I-N116L-A232V-Q245R,G020R-S024R-S078N-G097S-S101A-N116L-S128A,G020R-S024R-G097A-S101G-S128A-L217Q,T022R-S078N-S101G-S103A-V104I-N116L-A232V-Q245R,G020R-S024G-N043R-S078N-S101Q-Q109G-N116L-S128A-Q206R-L217Q,G020R-S024R-S078N-G097S-S101Q-N116L-S128A,G020R-S024R-S078N-G097S-S101N-Q109N-N116L-S128A,G020R-S024R-S101N-Q109G-N116L, G020R-S024R-S101Q-N116L-S128A-L217Q,G020R-N043R-S078N-S101G-Q109G-N116L,G020R-S024G-N043R-S078N-G097S-S101G-S128A,T022R-S024G-S078N-S101N-S103A-V104I-S128A-A232V-Q245R,T022R-S024G-G097A-S101Q-S103A-V104I-Q109G-N116L-S128A-A232V-Q245R,T022R-S101G-S103A-V104I-A232V-P239G-Q245R-E271I,G020R-S024R-S101Q-S128A, G020R-S024R-G097A-S101N-Q109G-N116L,G020R-S024G-N043R-S078N-G097A-S101G-Q109G-N116L,G020R-S024R-G097S-S101Q-Q109N-N116L-L217Q,T022R-S024G-S078N-G097A-S101Q-S103A-V104I-A232V-Q245R,G020R-S024R-G097A-S101G-Q109N-S128A,G020R-S024R-G097S-S101A-Q109N-S128A,T022R-G097S-S101Q-S103A-V104I-Q109G-A232V-Q245R,G020R-S024R-S078N-G097A-S101N-S128A-L217Q,G020R-S024G-N043R-S078N-G097S-S101G-N116L,G020R-S024R-S078N-G097S-S101G-Q109N-N116L-L217Q,T022R-S024G-S078N-S101N-S103A-V104I-Q109N-N116L-S128A-A232V-Q245R,G020R-S024R-S078N-G097S-S101A-Q109N-S128A-L217Q,G020R-S024R-S078N-G097A-S101G-Q109N-N116L-L217Q,G020R-S024G-N043R-S078N-S101Q,G020R-S024R-S078N-G097A-S101Q-Q109G-N116L,G020R-N043R-G097A-S101N-N116L-S128A,G020R-S024R-S078N-S101G-Q109G-N116L-S128A-L217Q,G020R-S024R-S078N-S101Q-Q109G-N116L-S128A-L217Q,G020R-S024R-S078N-G097S-S101G-Q109N-N116L-S128A,G020R-S024R-S078N-G097S-S101N-Q109N-N116L,T022R-S101N-S103A-V104I-Q109N-N116L-S128A-A232V-Q245R,T022R-S024G-G097S-S101G-S103A-V104I-Q109G-N116L-S128A-A232V-Q245R,T022R-S024G-S078N-S101G-S103A-V104I-N116L-S128A-A232V-Q245R,G020R-S024R-S078N-S101N-Q109N-N116L,G020R-S024R-S078N-S101A-Q109N-N116L-S128A,T022R-S024G-G097A-S101Q-S103A-V104I-N116L-S128A-A232V-Q245R,T022R-G097S-S101G-S103A-V104I-S128A-A232V-Q245R,T022R-G097A-S101Q-S103A-V104I-S128A-A232V-Q245R,T022R-S024G-S078N-G097S-S101Q-S103A-V104I-S128A-A232V-Q245R,T022R-S078N-G097A-S101Q-S103A-V104I-N116L-S128A-A232V-Q245R,G020R-S024R-S101G-Q109G-N116L-L217Q,G020R-S024R-S078N-S101Q-Q109N-N116L,G020R-S024R-S101G-N116L-S128A-L217Q,G020R-S024R-S101A-Q109G-N116L-S128A,T022R-S024G-S078N-G097A-S101G-S103A-V104I-N116L-L217Q-A232V-Q245R,T022R-S024G-S078N-G097S-S101Q-S103A-V104I-Q109G-N116L-S128A-A232V-Q245R,G020R-N043R-G097A-S101N-S128A-L233I,T022R-S101Q-S103A-V104I-A232V-Q245R,G020R-N043R-G097S-S101Q-Q109G-N116L-S128A,T022R-S078N-S101Q-S103A-V104I-A232V-Q245R,G020R-S024R-G097S-S101N-Q109G-N116L,T022R-S078N-G097S-S101G-S103A-V104I-S128A-A232V-Q245R,T022R-S024G-S101N-S103A-V104I-A232V-Q245R,G020R-S024R-G097A-S101N-Q109G-S128A,G020R-N043R-S101Q-N116L-S128A-L217Q,G020R-S024R-S078N-G097A-S101Q-Q109N-S128A,G020R-S024R-E089D-S101A-Q109N-N116L,G020R-S024R-G097A-S101G-Q109G-S128A,G020R-S024G-N043R-S101G-N116L-S128A-L217Q,G020R-N043R-S078N-S101Q-Q109G-L217Q,G020R-S024G-N043R-G097A-S101N-Q109N-N116L-L217Q,G020R-S024R-S078N-G097A-S101G-N116L-S128A,G020R-S024R-G097S-S101A-Q109N-N116L,G020R-S024R-S078N-S101A-Q109G-S128A,G020R-S024R-G097A-S101A-Q109G-N116L-S128A,G020R-N043R-G097A-S101Q-N116L-S128A,G020R-N043R-G097S-S101Q-S128A-L217Q,G020R-S024R-S078N-G097A-S101G-Q109G-N116L-L217Q,G020R-N043R-G097A-S101G-Q109N-N116L-S128A,G020R-N043R-S078N-G097S-S101N-Q109N-L217Q,G020R-S024R-G097S-A098E-S101A-N116L-S128A,G020R-S024R-S078N-G097S-S101A-Q109G-S128A,T022R-S024G-S078N-G097S-S101G-S103A-V104I-Q109N-N116L-L217Q-A232V-Q245R,G020R-S024R-S078N-G097A-S101Q-Q109N-N116L-L217Q,T022R-S024G-G097S-S101Q-S103A-V104I-S128A-A232V-Q245R,G020R-S024R-S101A-Q109N-N116L-S128A-L217Q,G020R-S024R-S101G-Q109G-N116L-S128A-L217Q,T022R-S078N-G097A-S101N-S103A-V104I-S128A-A232V-Q245R,G020R-S024G-N043R-S101N-N116L-S128A-L217Q,G020R-S024R-S078N-G097S-S101A-Q109N-N116L,G020R-S024R-S078N-G097A-S101N-Q109N-N116L-S128A,G020R-S024R-S078N-S101Q-Q109N-N116L-L217Q,T022R-S024G-S078N-S101N-S103A-V104I-N116L-S128A-A232V-Q245R,G020R-N043R-S101G-Q109N-N116L-S128A-L217Q,T022R-S101N-S103A-V104I-L217Q-A232V-Q245R,T022R-G097A-S101N-S103A-V104I-N116L-S128A-A232V-Q245R,G020R-S024R-G097A-S101A-S128A-L217Q,T022R-S024G-S078N-G097A-S101N-S103A-V104I-S128A-L217Q-A232V-Q245R,G020R-S024R-S078N-G097S-S101N-Q109G-N116L-S128A-L217Q,T022R-S024G-S101G-S103A-V104I-N116L-L217Q-A232V-Q245R,T022R-G097A-S101Q-S103A-V104I-N116L-S128A-A232V-Q245R,G020R-N043R-S101Q-S128A-L217Q,G020R-S024R-S078N-S101N-Q109G-S128A-L217Q,G020R-S024R-S078N-G097A-S101N-Q109N-N116L-S128A-L217Q,T022R-S024G-G097A-S101Q-S103A-V104I-N116L-L217Q-A232V-Q245R,G020R-S024R-S078N-G097S-S101G-S128A-N204S-L217Q,T022R-S024G-S078N-S101Q-S103A-V104I-N116L-S128A-A232V-Q245R,G020R-S024R-S078N-G097S-S101Q-Q109N-N116L-L217Q,G020R-S024R-S078N-S099G-S101Q-Q109N-N116L-L217Q,T022R-S024G-G097A-S101G-S103A-V104I-Q109G-N116L-L217Q-A232V-Q245R,T022R-S078N-G097A-S101Q-S103A-V104I-S128A-L217Q-A232V-Q245R,T022R-G097A-S101G-S103A-V104I-Q109G-N116L-L217Q-A232V-Q245R,G020R-S024R-G097A-S101N-Q109N-S128A-L217Q,T022R-G097S-S101N-S103A-V104I-S128A-L217Q-A232V-Q245R,G020R-S024R-K094R-S101N-N116L-S128A-L217Q,T022R-S024G-S078N-G097A-S101G-S103A-V104I-N116L-S128A-L217Q-A232V-Q245R,G020R-S024R-G097A-S101A-N116L-S128A-L217Q,G020R-S024R-S078N-G097W-S101Q-Q109N-N116L-L217Q,T022R-G097S-S101N-S103A-V104I-N116L-S128A-L217Q-A232V-Q245R,T022R-S101Q-S103A-V104I-N116L-L217Q-A232V-Q245R,T022R-S024G-S078N-G097A-S101Q-S103A-V104I-N116L-S128A-L217Q-A232V-Q245R,T022R-S078N-G097A-S101Q-S103A-V104I-Q109N-N116L-S128A-L217Q-A232V-Q245R,G020R-N043R-G097S-S101Q-Q109G-N116L-S128A-L217Q,T022R-S024G-G097A-S101G-S103A-V104I-N116L-S128A-L217Q-A232V-Q245R,T022R-G097S-S101G-S103A-V104I-L217Q-A232V-Q245R,T022R-S024G-G097A-S101Q-S103A-V104I-Q109G-N116L-L217Q-A232V-Q245R,T022R-S024G-S078N-G097A-S101Q-S103A-V104I-S128A-L217Q-A232V-Q245R,T022R-S101Q-S103A-V104I-Q109G-N116L-S128A-L217Q-A232V-Q245R,T022R-S078N-G097S-S101G-S103A-V104I-N116L-S128A-L217Q-A232V-Q245R,T022R-S024G-T071A-S078N-S101N-S103A-V104I-Q109N-N116L-S128A-A232V-Q245R,T022R-S024G-G097A-S101Q-S103A-V104I-S128A-L217Q-A232V-Q245R,T022R-S101G-S103A-V104I-Q109N-S128A-A232V-Q245R,T022R-S024G-G097S-S101G-S103A-V104I-S128A-L217Q-A232V-Q245R,T022R-S024G-S078N-S101Q-S103A-V104I-Q109N-N116L-S128A-L217Q-A232V-Q245R,T022R-S101G-S103A-A232V-Q245R,T022R-S101G-S103A-V104I-A232V-Q245R-E271A,T022R-S103A-V104I-A232V-Q245R, T022R-S101G-V104I-A232V-Q245R,T022R-S101G-S103A-V104I-Q245R, G020R-N043R-E271A,G020R-S024R-I035T-S101A-N116L, G020R-N043R-P239G, G020R-N043R-S242L, orG020R-N043R-V234F (List 17), and wherein amino acid positions of thesubtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO: 1.

The present invention further provides an isolated subtilisin variant,wherein said subtilisin variant is a mature form having proteolyticactivity and comprises an amino acid sequence comprising a combinationof amino acid substitutions at positions selected fromT022Y-S101N-S103A-V104I-A232T-Q245R,G020R-R045T-S101A-P210I-G211Q-L267F,T022Y-S101N-S103N-V104L-A232M-Q245R,T022A-P052T-S101G-S103A-V104I-L217Q-A232V-Q245R,T022A-S101G-S103N-V104L-A232T-Q245R,T022Y-S101A-S103N-V104L-A232V-Q245R,T022A-S101A-S103N-V104I-A232T-Q245R,T022Y-S101A-S103N-V104I-A232L-Q245R,T022Y-S101G-S103A-V104I-A232T-Q245R,T022A-S024G-N076D-S078N-G097S-S101N-S103A-V104I-L217Q-A232V-Q245R,T022A-S101N-S103N-V104L-A232M-Q245R,T022A-S101Q-S103A-V104I-S128A-P129S-A232V-Q245R,T022A-S101N-S103A-V104I-S128A-A232V-Q245R,S024R-S078D-G118S-G159D-Q245R-H249R,T022K-S101N-S103A-V104L-A232T-Q245R,T022R-S101T-S103N-V104I-A232T-Q245S,T022Q-S101N-S103N-V104I-A232M-Q245R, S024R-S078G-G118D-Q245R-H249R,T022R-S101N-S103N-V104L-A232V-Q245R,T022A-N076D-S101Q-S103A-V104I-Q109G-A232V-Q245R,G020R-S024R-S078G-G118D-S188D-Q245R-N248D,T022Y-S101N-S103A-V104I-A232V-Q245R,T022Q-S101N-S103A-V104L-A232L-Q245R,T022Q-S101N-S103N-V104I-A232L-Q245R,T022A-G097S-S101N-S103A-V104I-S128A-L217Q-A232V-Q245R,T022R-S101N-S103N-V104I-A232T-Q245S,G020R-S078D-G118D-G159D-Q245R-H249R,T022R-S101N-S103A-V104L-A232M-Q245R,T022A-N076D-G097S-S101Q-S103A-V104I-Q109N-S128A-A232V-Q245R,T022R-S101G-S103N-V104L-A232T-Q245S,G020R-T022W-N043R-R045T-S101A-G211Q-T213A,T022Q-S101T-S103N-V104I-A232T-Q245R,G020R-S024R-N043R-S101A-N204D-T213A,T022A-S024G-S101Q-S103A-V104I-Q109G-A232V-Q245R,G020R-S024R-S078D-G118D-S188D-Q245R-N248D-H249R,S024R-N043R-S101A-P210I,T022A-N076D-S101G-S103A-V104I-A232V-Q245R-A270V,G020R-S024R-S078D-G118S-H249R, S024R-S078G-G118S,T022R-S101G-S103N-V104L-A232V-Q245S, S024R-N043K-R045T-S101A,T022A-N076D-S078N-S101N-S103A-V104I-A232V-Q245R-T274A,T022A-S024G-N076D-S078N-S101G-S103A-V104I-L217Q-A232V-Q245R,T022Q-S101T-S103G-V104I-A232M-Q245W,S024R-S078G-G118S-P129E-Q245R-H249R,T022A-S024R-N076D-S101G-S103A-V104I-A232V-Q245R, V026W,T022R-S101G-S103N-V104I-A232L-Q245W, G020R-S024R-S078D-G118S-N248D,G020R-R045T-S101A-N204S-P210I,G020R-S024R-S078G-G118D-G159D-N248D-H249R,T022Y-S101A-S103N-V104I-A232V-Q245R,T022A-S024G-S078N-G097A-S101N-S103A-V104I-L217Q-A232V-Q245R,S024R-S101A-P210I-G211Q, T022R-S101N-S103N-V104I-A232V-Q245S,T022A-S024G-G097A-S101N-S103A-V104I-Q109G-A232V-Q245R,T022A-G097A-S101N-S103A-V104I-A232V-Q245R,T022A-S024G-N076D-S101Q-S103A-V104I-Q109N-A232V-Q245R,G020R-S078G-G118S-H249R, S078G-G118S-Q245R-H249R,T022A-G097A-S101N-S103A-V104I-L217Q-A232V-Q245R,T022Y-S101T-S103G-V104I-A232M-Q245R,T022K-S101G-S103N-V104I-A232L-Q245R,T022A-S024G-G097S-S101N-S103A-V104I-A232V-Q245R,S024R-S078G-G118D-G159D-S188D-Q245R-H249R,T022Q-S101N-S103A-V104I-A232V-Q245R,G020R-S024R-S078G-G118D-G159D-Q245R-N248D,T022A-S024G-S101G-S103A-V104I-A232V-Q245R,T022Q-S101G-S103A-V104L-A232T-Q245R,T022Q-S101T-S103A-V104I-A232T-Q245R,T022A-N076D-S101N-S103A-V104I-S128A-A232V-Q245R,T022A-S024G-N076D-S078N-G097A-S101G-S103A-V104I-A232V-Q245R,T022A-S101G-S103A-V104L-A232V-Q245R,T022A-S024G-S078N-G097S-S101Q-S103A-V104I-S128A-A232V-Q245R,T022A-S024G-N076D-G097S-S101G-S103A-V104I-A232V-Q245R,S024R-N043R-S101A-G211Q-T213A,T022A-G097S-S101Q-S103A-V104I-A232V-Q245R,S024R-S078G-G118S-Q245R-N248D,G020R-T022W-S024R-N043R-S101A-N204S-P210I-G211Q,T022A-S024G-S078N-G097S-S101N-S103A-V104I-Q109G-S128A-A232V-Q245R,T022A-S101G-S103A-V104I-P129S-A232V-Q245R,T022R-S101T-S103G-V104I-A232T-Q245S,T022A-S024G-T072V-G097S-S101Q-S103A-V104I-S128A-L217Q-A232V-Q245R,T022K-S101A-S103A-V104I-A232V-Q245W,G020R-T022W-S024R-R045T-S101A-N116L-P210I,T022R-S101T-S103G-V104I-A232I-Q245W,T022A-S024G-N076D-S078N-G097S-S101N-S103A-V104I-S128A-A232V-Q245R,T022A-G097A-S101N-S103A-V104I-S128A-A232V-Q245R,T022Y-S101T-S103N-V104L-A232V-Q245R,G020R-S024R-N062E-S078G-G118S-N248D,T022Q-S101G-S103A-V104I-A232V-Q245R,T022A-N076D-S078N-S101N-S103A-V104I-S128A-A232V-Q245R,T022A-S024G-G097A-S101N-S103A-V104I-S128A-A232V-Q245R,T022A-S024G-N076D-S078N-S101Q-S103A-V104I-A232V-Q245R,T022A-N076D-S078N-S101G-S103A-V104I-L217Q-A232V-Q245R,T022A-S024G-N076D-S101N-S103A-V104I-A232V-Q245R,T022R-S101N-S103A-V104I-A232L-Q245S,T022A-S078N-S101N-S103A-V104I-Q109N-L217Q-A232V-Q245R,T022R-S101A-S103A-V104I-A232V-Q245W,T022A-S101Q-S103A-V104I-A232V-Q245R,T022A-S078N-G097S-S101N-S103A-V104I-A232V-Q245R,T022A-G097A-S101Q-S103A-V104I-P129S-A232V-Q245R,T022A-S101T-S103A-V104I-A232L-Q245R,T022A-A085T-G097S-S101Q-S103A-V104I-L217Q-A232V-Q245R,T022A-S101T-S103N-V104L-S105G-A232M-Q245R,T022A-S101A-S103A-V104I-A232M-Q245S,T022Q-S101A-S103A-V104I-A232M-Q245R,T022Q-S101G-S103A-V104L-A232M-Q245R,T022A-S024G-G097A-S101Q-S103A-V104I-S128A-A232V-Q245R,T022A-S024G-G097S-S101Q-S103A-V104I-L217Q-A232V-Q245R,T022A-N076D-S078N-G097S-S101G-S103A-V104I-S128A-A232V-Q245R,T022A-G097A-S101G-S103A-V104I-H120Q-S128A-A232V-Q245R,T022A-G097A-S101N-S103A-V104I-S128A-P129S-L217Q-A232V-Q245R,G020R-S024R-N062E-S078G-G118D-Q245R,T022K-S101G-S103G-V104I-A232V-Q245R, S078G-G118S-S188D-Q245R-H249R,T022W-N043R-S101A-G211Q-T213A,T022A-S024G-N076D-G097S-S101N-S103A-V104I-Q109N-A232V-Q245R,T022Y-S101A-S103N-V104L-A232M-Q245R,G020R-T022W-S101A-N116L-G211Q-T213A,T022W-S024R-N043R-R045T-S101A-T213A, N043R-S101A-G211Q-T213A,T022Y-S101N-S103N-V104L-A232L-Q245R, T255G,T022A-G097S-S101N-S103A-V104I-S128A-A232V-Q245R,T022W-S024R-N043R-S101A-P210I-T213A, N269K,T022R-S101I-S103A-V104I-A232L-Q245S,T022A-S024G-T072V-S078N-G097S-S101N-S103A-V104I-S128X-A232V-Q245R,T022K-S101T-S103G-V104I-A232M-Q245R,H017R-N043R-R045T-S101A-P210I-T213A,T022A-S024G-G097S-S101G-S103A-V104I-S128A-A232V-Q245R,T022A-S078N-G097A-S101Q-S103A-V104I-A232V-Q245R,T022A-N076D-G097A-S101N-S103A-V104I-A232V-Q245R,T022A-S024G-V026A-S101G-S103A-V104I-S128A-A232V-Q245R,T022A-S024G-G097A-S101G-S103A-V104I-S128A-L217Q-A232V-Q245R,T022Q-S101T-S103G-V104I-A232L-Q245R,T022W-S024R-S101A-P210I-G211Q-T213A,T022Q-S101N-S103A-V104I-A232M-Q245R, G020R-R045T-S101A,T022Y-S101N-S103N-V104I-A232V-Q245R,T022R-S101G-S103A-V104I-A232L-Q245R,S024R-S078D-G118D-P129E-Q245R-H249R,T022R-S101A-S103A-V104I-A232M-Q245S, G020R-S101A-P210I-G211Q,T022W-N043R-R045T-S101A-P210I-G211Q,T022R-S101G-S103A-V104I-A232M-Q245S,T022A-S024G-N076D-S101G-S103A-V104I-L217Q-A232V-Q245R,T022R-S101T-S103N-V104I-A232V-Q245S,T022Q-S101G-S103N-V104L-A232M-Q245R,T022K-T038I-S101G-S103A-V104I-A232T-Q245S,N043R-R045T-S101A-P210I-G211Q, T022K-S101T-S103A-V104L-A232M-Q245R,T022Y-S101T-S103N-V104I-A232I-Q245R,G020R-S024R-N043R-S101A-P210I-T213A, S099G-S101G,T022A-S101N-S103N-V104L-A232T-Q245R,T022A-S024G-N076D-S078N-G097A-S101N-S103A-V104I-A232V-Q245R,T022R-S101N-S103A-V104I-A232V-Q245W, T022W-N043R-R045T-S101A-P210I,T022K-S101A-S103G-V104I-A232I-Q245R,T022Q-S101N-S103A-V104I-A232T-Q245S,T022A-S101Q-S103A-V104I-Q109G-A232V-Q245R,T022A-G097A-S101Q-S103A-V104I-S128A-P129S-L217Q-A232V-Q245R,T022A-S078N-G097A-S101Q-S103A-V104I-L217Q-A232V-Q245R,S024R-S078G-G118S-G159D-Q245R, T022K-S101N-S103N-V104I-A232T-Q245R,A098F-S099A-V104I, G020R-R045T-S101A-G211Q-T213A,T022A-N076D-S078N-G097A-S101G-S103A-V104I-A232V-Q245R,S024R-S078D-G118S-G159D-Q245R,T022A-S024G-N076D-S101N-S103A-V104I-Q109N-A232V-Q245R,T022K-S101T-S103N-V104I-A232M-Q245R,T022A-S024G-S078N-G097S-S101N-S103A-V104I-S128A-A232V-Q245R,T022Q-S101T-S103N-V104I-A232L-Q245S, A114T,T022Q-S101N-S103N-V104L-A232I-Q245R,T022A-S078N-S101G-S103A-V104I-Q109N-A232V-Q245R,T022K-S101I-S103A-V104I-A232M-Q245R,T022A-S024G-G097S-S101Q-S103A-V104I-Q109G-S128A-L217Q-A232V-Q245R,T022A-S101G-S103A-V104I-N116L-Y209V-G211Q-A232V,T022A-S101T-S103N-V104I-A232M-Q245W,G020R-T022W-S024R-S101A-N116L-G211Q,T022A-S078N-S101Q-S103A-V104I-S128A-L217Q-A232V-Q245R,G020R-R045T-S101A-G211Q,T022A-N076D-S078N-G097A-S101G-S103A-V104I-S128A-A232V-Q245R,S024R-S101A-T213A, T022A-S078N-S101G-S103A-V104I-A232V-Q245R,G020R-S024R-S078D-G118S-G159D-S188D-Q245R-N248D-H249R,T022Y-S101N-S103N-V104I-A232V-Q245W,T022Y-S101A-S103G-V104I-A232V-Q245R,T022A-S024R-N076D-S101G-S103A-V104I-A232V-N238Y-Q245R,G020R-S078G-G118S-G159D-H249R,T022A-S024R-S101G-S103A-V104I-P129E-A232V-Q245R-A272V,T022R-S101N-S103A-V104I-A232V-Q245S, G020R-S101A-P210I-G211Q-T213A,G020R-S024R-S078D-G118S-G159D-S188D-Q245R, S087R,G020R-S024R-R045T-A048T-S101A-G211Q,T022A-S024G-G097S-S101Q-S103A-V104I-A232V-Q245R,G020R-S024R-S078G-G118S-P129E-S188D-Q245R-N248D-H249R,T022A-G097S-S101G-S103A-V104I-Q109N-A232V-Q245R,L021S-T022A-S024G-N076D-S078N-S101G-S103A-V104I-S128A-A232V-Q245R,S242K, T022K-S101A-S103A-V104I-A232V-Q245R,T022K-S101N-S103A-V104L-A232L-Q245R,T022A-N076D-S101G-S103A-V104I-A232V-N238Y-Q245R-A270V-A272V,T022R-S101T-S103A-V104I-A232V-Q245S,T022Y-S101N-S103G-V104I-A232L-Q245R,T022Q-S101N-S103G-V104I-A232V-Q245R,T022A-S101G-S103A-V104I-A232V-Q245W,T022R-S101A-S103A-V104I-A232L-Q245S,G020R-T022W-R045T-F050L-S101A-P210I-G211Q-T213A,T022A-S101Q-S103A-V104I-S128A-L217Q-A232V-Q245R,G020R-S101A-G211Q-T213A,T022A-N076D-S101G-S103A-V104I-A232V-Q245R-A272V,T022A-S101G-S103A-V104I-A232V-N238Y-Q245R-A270V,T022A-S101N-S103G-V104I-A232V-Q245R, G020R-S101A-T208P-P210I-G211Q,D181S, S101G-S103A-V104I-N116L-Y209V-A232V,T022Q-S101A-S103N-V104I-A232I-Q245R,T022A-S101G-S103N-V104L-G211S-A232T-Q245R,T022A-G097A-S101Q-S103A-V104I-L217Q-A232V-Q245R,T022A-S101T-S103N-V104I-A232M-Q245S,T022A-S101G-S103A-V104I-Q109N-A232V-Q245R,T022Q-S101A-S103G-V104I-A232M-Q245R,G020R-S078G-G118S-G159D-N248D-H249R,T022A-N076D-S101N-S103A-V104I-A232V-Q245R,T022A-S024G-G097S-S101N-S103A-V104I-Q109G-S128A-A232V-Q245R,T022Q-S101A-S103A-V104I-A232T-Q245R,T022A-S101G-S103A-V104I-A232L-Q245R,T022A-S101G-S103A-V104I-P129E-A232V-N238Y-V244I-Q245R-A270V-A272V,G020R-S024R-S078D-G118D-G159D-Q245R-N248D-H249R,T022R-S101A-S103N-V104I-A232V-Q245W,T022Q-S101A-S103N-V104I-A232M-Q245R,T022Q-S101A-S103N-V104L-A232L-Q245R,T022A-S024G-S101Q-S103A-V104I-S128A-L217Q-A232V-Q245R,T022Q-S101G-S103G-V104I-A232M-Q245R,G020R-S078G-G118S-G159D-Q245R-H249R,T022R-S101A-S103N-V104L-A232V-Q245S,T022A-S024V-G097S-S101N-S103A-V104I-H120N-S128A-A232V-Q245R,T022A-A073V-N076D-S078N-S101N-S103A-V104I-A232V-Q245R,T022Q-S101G-S103N-V104L-A232V-Q245R-T274I,T022A-S101N-S103N-V104I-A232T-Q245R,T022W-S024R-R045P-S101A-P210I-G211Q-T213A,G020R-S024R-N062E-S078G-G118D-Q245R-N248D,T022Q-S101A-S103N-V104L-A232M-Q245R,T022Q-S101I-S103G-V104L-A232T-Q245R, T022R-S101G-S103N-A232L-Q245R,T022A-G097S-S101Q-S103A-V104I-Q109N-L217Q-A232V-Q245R,T022A-S024G-G097A-S101Q-S103A-V104I-S128A-L217Q-A232V-Q245R,S024R-S101A-G211Q-T213A, T022R-S101N-S103N-V104L-A232T-Q245S,G020R-R045T-S101A-P210I, T022R-S101A-S103A-V104I-A232M-Q245W,T022A-S101N-S103N-V104I-A232L-Q245R,T022A-S101G-S103G-V104I-A232M-Q245R,G020R-S024R-S078D-G118S-S188D-Q245R-N248D, S099T-S103G,T022W-N043R-S101A-P210I, T022W-A029V-N043R-S101A-P210I, F050R,T022A-S024G-N076D-S078N-G097S-S101Q-S103A-V104I-Q109N-A232V-Q245R,T022A-S024G-N076D-S101G-S103A-V104I-A232V-Q245R,T022R-S101G-S103N-V104L-A232L-Q245R,T022A-S101N-S103A-V104I-Q109N-S128A-A232V-Q245R, S024R-R045T-S101A,T022A-N076D-G097S-S101N-S103A-V104I-L217Q-A232V-Q245R,T022Q-S101G-S103A-V104I-A232M-Q245S,T022W-S024R-N043R-R045T-S101A-G211Q-T213A, G020R-S078G-G118S,T022W-N043R-G046C-S101A-P210I-G211Q, S101A-G211Q-T213A,T022A-N076D-S078N-S101G-S103A-V104I-A232V-Q245R,T022A-S024G-N076D-G097S-S101Q-S103A-V104I-A232V-Q245R,T022A-S101N-S103A-V104I-L126I-P129S-A232V-Q245R,T022R-S101T-S103N-V104I-A232L-Q245W,T022A-S101G-S103A-V104I-A232V-N238Y-Q245R-A270V-A272V,S078G-G118D-Q245R,T022A-S024R-S101G-S103A-V104I-P129E-A232V-N238Y-Q245R-A270V,T022A-N076D-G097S-S101N-S103A-V104I-Q109G-S128A-A232V-Q245R,T022W-S024R-R045T-S101A-N116L-P210I-T213A,T022R-S101T-S103N-V104I-A232L-Q245S,T022W-S024R-N043R-R045T-S101A-S106L-P210I-G211Q,T022A-E089G-G097A-S101Q-S103A-V104I-P129S-L217Q-A232V-Q245R,T022A-S101A-S103G-V104I-A232L-Q245S, N043R-S101A-P210I-T213A,N043R-R045T-S101A-T213A, S078G-G118D-H120N-H249R,T022Q-S101T-S103N-V104I-A232L-Q245R-R275H,T022Y-S101A-S103A-V104I-A232T-Q245S.T022K-S101N-S103N-V104I-A232M-Q245R,T022R-S101T-S103N-V104L-A232V-Q245S,T022A-N076D-G097S-S101N-S103A-V104I-A232V-Q245R, M119L,A098F-S099A-S101G-V104I, S078D-G118S-Q245R,T022Y-S101N-S103A-V104I-A232L-Q245S,T022A-G097A-S101N-S103A-V104I-S128A-L217Q-A232V-Q245R,G020R-S078G-G118D-N248D-H249R, T022R-S101A-S103N-V104I-A232T-Q245S,T022R-S101G-S103N-V104I-A232L-Q245S,S024R-S078D-G118D-S188D-Q245R-H249R, S132H, A098Q-S099G-S101G-V104L,N043R-S101A-P210I-G211Q-T213A, T022Q-S101G-S103A-V104I-A232M-Q245W,T022A-S101Q-S103A-V104I-L126I-P129S-A232V-Q245R,T022K-S101T-S103G-V104I-A232M-Q245S,T022W-N043R-R045T-S101A-G211Q-T213A,T022Q-S101N-S103G-V104I-A232V-Q245W, DI 81T,G020R-S024R-S078D-G118S-P129E-G159D-Q245R-H249R,G020R-S024R-S078G-G118D-G159D-S188D-Q245R,T022A-S101T-S103N-V104L-A232L-Q245S,T022A-S101G-S103N-V104L-A232V-Q245S,T022A-S024G-G097A-S101N-S103A-V104I-S128A-L217Q-A232V-Q245R,T022A-S024G-N076D-S101N-S103A-V104I-S128A-A232V-Q245R,T022Y-S101T-S103A-V104L-A232M-Q245R,T022Q-S101T-S103N-V104L-A232T-Q245R, F050S,T022Y-S101G-S103N-V104I-A232V-Q245R, G020R-R045T-S101A-T213A,T022K-S101N-S103G-V104I-A232V-Q245W,S024R-S078G-G118D-P129E-G159D-Q245R-H249R,T022Q-S101T-S103N-V104I-A232M-Q245W, S049W,T022A-S101A-S103N-V104L-A232L-Q245R,T022A-N076D-S101G-S103A-V104I-I107V-P129G-A232V-Q245R-A270V,S099A-V104I, T022Q-S101A-S103G-V104I-A232V-Q245W,T022A-N076D-S101G-S103A-V104I-A232V-N238Y-Q245R,T022A-S101G-S103A-V104I-L124V-L217Q-A232V-Q245R,T022R-G097S-S101Q-S103A-V104I-S128A-L217Q-A232V-Q245R,T022R-G097A-S101Q-S103A-V104I-Q109N-S128A-A232V-Q245R,R010A-T022R-S024F-S101G-S103A-V104I-A232V-Q245R-L267N-E271H,G020R-S024G-N043R-S078N-S101G-Q109N-N116L-L217Q-M222S,G020R-N043R-E271H, G020R-N043R-E271I, G020R-S024R-S101A-N116L-N269I,T022R-S101G-S103A-V104I-A232V-Q245R-E271F, G020R-N043R-E271F,G020R-N043R-N269I, G020R-S024R-S101A-N116L-E271H,T022R-S101G-S103A-V104I-A232V-Q245R-E271H, G020R-I035T-N043R,G020R-S024R-S101A-A114T-N116L, G020R-S024R-S101A-N116L-E271I,G020R-N043R-A114T, G020R-N043R-L267N, G020R-S024R-S101A-N116L-L267N,G020R-S024R-V028A-S101A-N116L, G020R-S024R-S101A-N116L-G258R,G020R-N043R-G258R, G020R-S024R-S101A-N116L-S242L,G020R-S024R-S101A-N116L-P239G, I008N-G020R-S024R-S101A-N116L,G020R-S024R-S101A-N116L-V234F, T022R-S101G-S103A-V104I-A232V,G020R-S024R-S101A-N116L-E271A, R010A-G020R-N043R,G020R-S024R-S101A-N116L-E271F, G020R-V028A-N043R, 1008N-G020R-N043R, orG020R-S024F-S101A-N116L (List 18), and wherein amino acid positions ofthe subtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention further provides an isolated subtilisin variant,wherein said subtilisin variant is a mature form having proteolyticactivity and comprises an amino acid sequence comprising a combinationof amino acid substitutions at positions selected fromG020R-S024R-S078D-G118D-G159D-H249R,G020R-S024R-S078G-G118S-G159D-S188D-Q245R-N248D-H249R,T022A-S101T-S103G-V104I-A232T-Q245S,T022A-S101T-S103G-V104I-A232T-Q245R,T022Q-S101G-S103N-V104I-A232V-Q245W, T022W-N043R-S101A-T213A,T022A-N076D-S078N-G097A-S101N-S103A-V104I-S128A-A232V-Q245R,T022A-S024G-S078N-S101Q-S103A-V104I-S128A-A232V-Q245R,T022K-S101N-S103N-V104L-A232T-Q245R,T022A-S024G-S101Q-S103A-V104I-S128A-A232V-Q245R,G020R-S024R-N062E-S078G-G118D, G020R-S078G-G118D,T022Y-S101T-S103G-V104L-A232T-Q245R,R010H-T022A-S101G-S103A-V104I-A232V-Q245R-A272V,T022A-S024R-S101G-S103A-V104I-P129E-A232V-Q245R-A270V-A272V,T022A-N076D-S078N-G097S-S101Q-S103A-V104I-Q109G-A232V-Q245R,G020R-S024R-S078D-G118S-P129E-Q245R,T022A-N076D-S101G-S103A-V104I-A232V-N238Y-Q245R-A272V,T022K-S101N-S103N-V104I-A232V-Q245W,T022Q-S101T-S103N-V104I-A232M-Q245S,T022A-S078N-S101N-S103A-V104I-Q109G-L217Q-A232V-Q245R, W113Y,T022A-S101A-S103N-V104I-A232M-Q245S,T022A-S101Q-S103A-V104I-S128A-A232V-Q245R,G020R-S024R-S078G-G118D-G159D-S188D-H249R,T022A-S101N-S103G-V104I-A232T-Q245R,T022K-S101G-S103G-V104I-A232L-Q245R,T022A-N076D-G097A-S101Q-S103A-V104I-S128A-A232V-Q245R,T022Q-S101T-S103N-V104I-A232V-Q245S,T022A-S024F-S101G-S103A-V104I-N116L-Y209V-T213A-A232V,T022A-S101N-S103G-V104L-A232T-Q245R,T022A-S024R-S101G-S103A-V104I-P129E-A232V-N238Y-Q245R,T022A-N076D-G097S-S101Q-S103A-V104I-A232V-Q245R,T022A-N076D-S078N-G097A-S101N-S103A-V104I-A232V-Q245R,T022A-S101N-S103A-V104I-Q109G-S128A-A232V-Q245R, S259W,A016S-T022A-S024F-S101G-S103A-V104I-N116A-Y209V-G211Q-A232V,T022W-S101A-P210I-G211Q-T213A,T022A-S024G-N076D-G097S-S101N-S103A-V104I-A232V-Q245R, V028T-A215V,S024R-S078D-G118S-S188D-Q245R, T022Q-S101T-S103A-V104I-A232V-Q245W,T022A-S101N-S103A-V104I-Q109G-L217Q-A232V-Q245R,T022A-S024G-T071A-G097S-S101Q-S103A-V104I-S128A-A232V-Q245R, H120P,G020R-S024R-N062E-S078D-G118D-Q245R,T022A-N076D-S101Q-S103A-V104I-A232V-Q245R,T022Q-S101A-S103N-V104I-A232L-Q245W,T022Q-S101N-S103A-V104L-A232T-Q245R,T022W-S024R-N043R-R045T-S101A-N116L-P210I-G211Q,T022A-G097S-S101Q-S103A-V104I-S128A-L217Q-A232V-Q245R,G020R-S078G-G118S-G159D-S188D-H249R, A013S, A232N,T022A-N076D-S101G-S103A-V104I-A232V-Q245R,T022A-S024G-N076D-S078N-G097S-S101N-S103A-V104I-A232V-Q245R, I035T,V051M, N123A, G020R-S078G-G118S-G159D-Q245R-N248D,T022Y-S101A-S103N-V104I-A232L-Q245W,T022A-S024R-S101G-S103A-V104I-P129E-A232V-Q245R-A270V,G020R-S078D-G118S, T022R-S101N-S103N-V104L-A232M-Q245W,T022A-S024G-N076D-G097S-S101G-S103A-V104I-S128A-L217Q-A232V-Q245R,T022A-S101G-S103N-V104L-A232V-Q245R,T022A-N076D-G097A-S101Q-S103A-V104I-A232V-Q245R,T022A-S024R-N076D-S101G-S103A-V104I-I107V-P129E-A232V-Q245R,S024R-R045T-S101A-P210I-G211Q,T022A-S078N-S101N-S103A-V104I-Q109N-S128A-A232V-Q245R,T022A-S024G-S078N-G097A-S101N-S103A-V104I-Q109N-A232V-Q245R,G020R-S024R-S078G-G118D-P129E-G159D-H249R,G020R-S078G-G118D-P129E-S188D-Q245R-H249R, PO 14W,G020R-S024R-S078G-G118S-G159D-S188D-Q245R-N248D,T022R-S101G-S103N-V104I-A232M-Q245W,T022R-A098V-S101G-S103N-V104L-A232T-Q245R,T022A-S101I-S103A-V104I-A232V-Q245W, G020R-T022W-R045T-S101A-T213A,N043R-R045T-S101A, T022K-S101N-S103A-V104I-A232V-Q245R,T022K-S101T-S103N-V104I-A232L-Q245S, S056V,G020R-T022W-R045T-S101A-G211Q, G020R-S024R-S078D-G118D-Q245R-N248D,G020R-S024R-S078D-G118S-G159D-V244L-Q245R-N248D,T022A-G097A-S101Q-S103A-V104I-Q109G-S128A-A232V-Q245R,T022A-N076D-G097S-S101G-S103A-V104I-A232V-Q245R,G020R-T022W-R045T-S101A-N116L-P210I,T022Q-S101N-S103N-V104I-A232M-Q245S,T022Y-S101G-S103N-V104I-A232V-Q245W, A114V, S141C,T022A-N076D-S078N-S101N-S103A-V104I-A232V-Q245R, S056H,S078D-G118D-Q245R, S024R-N043R-R045T-S101A-T213A,T022Y-S101T-S103G-V104I-A232V-Q245S,T022A-S024G-G097S-S101Q-S103A-V104I-S128A-L217Q-A232V-Q245R, N269W,S078G-G118S-G159D-Q245R-N248D-H249R, T022W-S024R-R045T-S101A,T022A-S024R-S101G-S103A-V104I-P129E-A232V-N238Y-Q245R-A270V-A272V,T022A-S024R-S101G-S103A-V104I-P129E-A232V-Q245R,T022A-S024R-N076D-S101G-S103A-V104I-I107V-P129E-A232V-N238Y-Q245R-A270V-A272V,A122F, S024R-S078D-G118S-P129E-G159D-Q245R-H249R,T022K-S101T-S103G-V104I-A232V-Q245S,S009G-T022A-S078N-S101Q-S103A-V104I-Q109G-S128A-L217Q-A232V-Q245R,A098Q, T022A-S101N-S103A-V104L-A232L-Q245R,T022A-S101A-S103N-V104I-A232T-Q245W,T022A-S024G-N076D-G097A-S101N-S103A-V104I-A232V-Q245R,S024R-S078G-G118D-G159D-H249R,T022A-S024G-N076D-G097S-S101N-S103A-V104I-S128A-A232V-Q245R,T022A-S024G-N076D-S078N-S101Q-S103A-V104I-S128A-A232V-Q245R,T022A-S101Q-S103A-V104I-L124V-S128A-P129S-A232V-Q245R,T022R-S101N-S103A-V104I-A232M-W241R-Q245W, R045T-S101A-P210I-G211Q,G020R-S024R-S078G-G118S-P129E-Q245V-N248E,T022Q-S101T-S103N-V104I-A232L-Q245W,T022A-S024G-S078N-S101N-S103A-V104I-Q109G-L217Q-A232V-Q245R,T022A-S024G-G097A-S101N-S103A-V104I-L217Q-A223S-A232V-Q245R,T022K-S101T-S103A-V104I-A232T-Q245W, N043R-S101A-G211Q,T022A-S101G-S103A-V104I-Q109N-S128A-A232V-Q245R,T022A-G097A-S101G-S103A-V104I-S128A-L217Q-A232V-Q245R,G020R-S078G-G118S-P129E-H249R, S024R-S078G-G118S-G159D-S188D-Q245R,N043R-R045T-S101A-N204S-P210I-T213A,T022R-S101N-S103A-V104L-A232V-Q245S,T022R-S101N-S103G-V104I-A232V-Q245S,T022K-S101T-S103N-V104I-A232M-Q245S,T022A-N076D-S101N-S103A-V104I-L217Q-A232V-Q245R,T022A-S024G-N076D-S078N-S101N-S103A-V104I-Q109G-S128A-A232V-Q245R,G020R-T022W-R045T-S101A-P210I-T213A,T022A-N076D-G097A-S101G-S103A-V104I-L217Q-A232V-Q245R,T022W-S101A-P210I,T022A-G097A-S101N-S103A-V104I-L126I-P129S-A232V-Q245R,T022A-S078D-S101N-S103A-V104I-S128A-A232V-Q245R, Q236L,T022Y-S101T-S103N-V104I-A232V-Q245S, A200D,G020R-S078G-G118S-S188D-H249R, T022A-S101G-S103N-V104I-A232T-Q245W,T022A-S024G-N076D-G097A-S101N-S103A-V104I-S128A-A232V-Q245R, G157I,G020R-S024R-S078G-G118S-P129E-G159D-S188D-Q245R,T022Y-S101A-S103G-V104L-A232T-Q245R, S024R-S078G-G118D-H249R,T022Q-S101A-S103N-V104L-A232T-Q245S,T022Y-S101A-S103N-V104I-A232T-Q245W,T022A-S024G-S101N-S103A-V104I-Q109N-L217Q-A232V-Q245R,T022A-S101G-S103N-V104I-A232M-Q245S,T022A-S024G-N076D-S078N-S101G-S103A-V104I-S128A-A232V-Q245R,T022A-S101G-S103N-V104I-A232M-Q245W, I008N,T022A-S101N-S103G-V104I-A232M-Q245S, V026A,T022R-S101N-S103A-V104L-A232T-Q245W,T022A-S101A-S103N-V104I-A232L-Q245S,T022A-S101N-S103A-V104I-A232T-Q245R,N043R-R045T-S101A-P210I-G211Q-T213A,T022A-S024G-N076D-G097A-S101Q-S103A-V104I-S128A-A232V-Q245R,A098F-S099T-S101A, A098F-S099T-S101G-V104L, P014N, A098Q-S103G-V104I,S099T-S103A-V104I, S087W, S024R-N062E-S078G-G118S-H249R,S024R-N043R-R045T-S101A-G211Q, N043R-R045T-S101A-G211Q,G020R-S078G-G118S-N248D, T022Q-S101N-S103N-V104I-A232T-Q245W,S078D-G118S-P129E-Q245R-H249R, T022Q-S101T-S103G-V104I-A232T-Q245S,T022A-S024G-S101N-S103A-V104I-Q109N-S128A-A232V-Q245R,S024F-S101G-S103A-V104I-N116L-Y209A-T213A-A232V,S101A-N116L-P210I-T213A, T022R-S101N-S103N-V104L-A232L-Q245S, B015T,T022A-S101G-S103A-V104I-L124V-A232V-Q245R,G020R-S024R-S078D-G118D-G159D-Q245R, A114I, S212A,T022A-S101T-S103N-V104I-A232L-Q245S,T022A-S024G-S078N-G097S-S101N-S103A-V104I-S128A-T143I-L217Q-A232V-Q245R,S024R-S078G-G118S-G159D-S188D-Q245R-H249R,T022K-S101A-S103N-V104L-A232L-Q245S, T022W-S101A-T213A,T022A-N076D-S078N-G097S-S101Q-S103A-V104I-A232V-Q245R, S132V,S024R-S078G-G118D-S188D-Q245R-N248D-H249R, A098Q-S101A-S103A-V104L,A098Q-S099T-S103A, T022R-S101T-S103N-V104L-A232T-Q245W,T022A-N076D-S101Q-S103A-V104I-S128A-A232V-Q245R,V051A-S101A-P210I-T213A, A098Q-S099T-S101A-V104L,T022R-S101N-S103N-V104I-A232M-Q245W, S099A-S101A-S103A,G020R-S078G-G118D-S188D-Q245R,G020R-S024R-S078D-G118S-P129E-G159D-S188D-Q245R-H249R,T022A-N076D-S078N-G097A-S101Q-S103A-V104I-L217Q-A232V-Q245R,S024R-S078G-G118D, T022Q-S101N-S103N-V104L-A232M-Q245R,T022A-S101T-S103G-V104I-A232M-Q245S, A048G,T022Q-S099G-S101A-S103N-V104I-A232V-Q245R,S024R-R045T-S101A-G211Q-T213A,S024R-R045T-S101A-I107V-A114T-N116L-P210I-G211Q-T213A,T022R-S101T-S103G-V104I-A232L-Q245S, S099G-S103A, A098F-S099A,T022R-S101N-S103G-V104I-A232V-Q245W,T022A-S103A-V104I-L124T-A232V-Q245R,T022A-S024F-S101G-S103A-V104I-N116L-Y209V-A232V,T022K-S101N-S103N-V104L-A232V-Q245S,T022K-S101A-S103A-V104L-A232M-Q245R, T022Q-S101G-S103N-VI04I-A232T-Q245W, T022A-S101T-S103A-V104L-A232T-Q245W,T022A-S101N-S103N-V104I-A232V-Q245S,T022A-N076D-S078N-G097A-S101N-S103A-V104I-L217Q-A232V-Q245R, A098M,T022A-S101G-S103G-V104I-A232L-Q245R, G020R-S101A-S207G-P210I-T213A,T022A-S024G-G097A-S101Q-S103A-V104I-Q109G-S128A-A232V-Q245R,G020R-S024R-S078G-G118S-G159D-Q245R-N248D-H249R,G020R-S024R-S078D-G118S-P129E-G159D-Q245R,T022Y-S101G-S103N-V104I-A232L-Q245S, V026Q, S240G,G020R-S024R-S078G-G118S-S188D, G020R-S024R-S078G-G118D-N238D-N248D,T022R-S101G-S103A-V104I-A232T-Q245S,T022W-S024R-R045T-S101A-P210I-G211Q,T022A-S024G-N076D-S078N-G097S-S101Q-S103A-V104I-A232V-Q245R,T022W-S024R-R045T-S101A-T213A, A200F,T022R-S101T-S103G-V104L-A232T-Q245S,T022A-S078N-G097S-S101Q-S103A-V104I-Q109N-L217Q-A232V-Q245R,T022A-S101G-S103N-V104I-A232T-Q245S,T022A-S101I-S103N-V104I-A232T-Q245S,T022A-S101A-S103A-V104L-A232V-Q245S, S099G-S101A-V104L,H017R-T022A-S024V-N076D-G097S-S101N-S103A-V104I-Q109N-S128A-A232V-Q245R,T022A-S101A-S103N-V104L-A232M-Q245S,T022A-S101Q-S103A-V104I-Q109G-L217Q-A232V-Q245R,T022A-S024G-N076D-S101Q-S103A-V104I-S128A-A232V-Q245R,T022A-S024G-S078N-G097S-S101N-S103A-V104I-S128A-S212P-L217Q-A232V-Q245R,S056L, T253L, T022W-R045T-S101A-G211Q-T213A,T022A-G097A-S101Q-S103A-V104I-S128A-L217Q-A232V-Q245R,T022A-S024G-N076D-S078N-G097A-S101N-S103A-V104I-S128A-A232V-Q245R,G020R-T022W-R045T-S101A-G211Q-T213A, S101A-G211Q, N123D,T022R-S101A-S103N-V104I-A232M-Q245W,T022Y-S101T-S103N-V104L-A232I-Q245R,T022A-S101G-S103A-V104I-N116L-Y209V-G211Q-T213A-A232V,G020R-S024R-S078D-G118D-N248D, A098Q-S099G-S101A, S099T-V104I,T022Q-S101G-S103A-V104I-A232V-Q245S,T022R-S101A-S103G-V104I-A232T-Q245S,T022K-S101T-S103N-V104I-A232I-Q245W, A016M,T022A-S024G-N076D-S101N-S103A-V104I-Q109G-A232V-Q245R,T022K-S101N-S103N-V104I-A232T-Q245S,G020R-N043R-R045T-S101A-N204D-P210I-G211Q,T022A-S101I-S103N-V104I-A232V-Q245S,T022Y-S101G-S103N-V104L-A232V-Q245R,T022A-S101I-S103N-V104I-A232V-Q245W,T022A-S024G-S078N-G097S-S101N-S103A-V104I-Q109G-L217Q-A232V-Q245R,A088I, T022Y-S101A-S103A-V104I-A232M-Q245S,T022W-S024R-R045T-S101A-N116L-P210I-G211Q,T022W-N043R-R045T-S101A-P210I-G211Q-T213A, 1038A,S078D-G118S-G159D-Q245R, T022K-S101T-S103A-V104L-A232V-Q245W,T022A-S024G-N076D-S078N-G097S-S101N-S103A-V104I-Q109G-A232V-Q245R,T022A-S078N-S101Q-S103A-V104I-Q109N-L217Q-A232V-Q245R, G025S, G025I,A016S-S101G-S103A-V104I-N116L-M119V-Y209V-G211Q-A232V,T022A-N076D-V084A-S101G-S103A-V104I-A232V-Q245R, V011L,T022Y-S101T-S103G-V104I-A232M-Q245S,T022R-S101N-S103A-V104L-A232I-Q245S, S240V,G020R-S024R-S078G-G118D-P129E-S188D-H249R,T022W-S024R-R045T-S101A-G211Q-T213A,T022A-S024G-N076D-G097A-S101Q-S103A-V104I-Q109G-A232V-Q245R, V004Y,V051A, G195W-N269E, S024R-S078G-G118D-N248D-H249R,T022A-S099G-S101T-S103G-V104I-A232V-Q245S,T022Q-S101T-S103G-V104I-A232L-Q245W,T022Q-S101N-S103N-V104I-A232V-Q245W;T022A-S101T-S103G-V104I-A232V-Q245W,T022K-S101N-S103G-V104I-A232V-Q245R,T022Q-S101G-S103N-V104I-A232V-Q245S,T022R-S078N-G097S-S101G-S103A-V104I-Q109G-S128A-A232V-Q245R,T022R-S101G-S103A-V104I-M222S-A232V-Q245R-E271H,G020R-S024R-S101N-Q109G-N116L-M222S, R010A-G020R-S024R-S101A-N116L, orG020R-N043R-M222S (List 19), and wherein amino acid positions of thesubtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention further provides any of the above isolatedsubtilisin variants, wherein the subtilisin variant is a proteasevariant of Bacillus lentus subtilisin GG36 protease, wherein saidBacillus lentus subtilisin GG36 protease comprises the amino acidsequence shown in SEQ ID NO:2.

The present invention further provides an isolated subtilisin variant ofBacillus lentus subtilisin GG36 protease, wherein said Bacillus lentussubtilisin GG36 protease comprises the amino acid sequence shown in SEQID NO:2, and wherein said subtilisin variant has at least 80% amino acidsequence identity to SEQ ID NO:2 and has proteolytic activity andcomprises an amino acid sequence comprising a combination of amino acidsubstitutions selected from List 1, and wherein amino acid positions ofthe subtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention further provides an isolated subtilisin variant ofBacillus lentus subtilisin GG36 protease, wherein said Bacillus lentussubtilisin GG36 protease comprises the amino acid sequence shown in SEQID NO:2, and wherein said subtilisin variant has at least 80% amino acidsequence identity to SEQ ID NO:2 and has proteolytic activity andcomprises an amino acid sequence comprising a combination of amino acidsubstitutions selected from List 2, and wherein amino acid positions ofthe subtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention further provides an isolated subtilisin variant ofBacillus lentus subtilisin GG36 protease, wherein said Bacillus lentussubtilisin GG36 protease comprises the amino acid sequence shown in SEQID NO:2, and wherein said subtilisin variant has at least 80% amino acidsequence identity to SEQ ID NO:2 and has proteolytic activity andcomprises an amino acid sequence comprising a combination of amino acidsubstitutions selected from List 3, and wherein amino acid positions ofthe subtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention further provides an isolated subtilisin variant ofBacillus lentus subtilisin GG36 protease, wherein said Bacillus lentussubtilisin GG36 protease comprises the amino acid sequence shown in SEQID NO:2, and wherein said subtilisin variant has at least 80% amino acidsequence identity to SEQ ID NO:2 and has proteolytic activity andcomprises an amino acid sequence comprising a combination of amino acidsubstitutions selected from List 4, and wherein amino acid positions ofthe subtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention further provides an isolated subtilisin variant ofBacillus lentus subtilisin GG36 protease, wherein said Bacillus lentussubtilisin GG36 protease comprises the amino acid sequence shown in SEQID NO:2, and wherein said subtilisin variant has at least 80% amino acidsequence identity to SEQ ID NO:2 and has proteolytic activity andcomprises an amino acid sequence comprising a combination of amino acidsubstitutions selected from List 5, and wherein amino acid positions ofthe subtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention further provides an isolated subtilisin variant ofBacillus lentus subtilisin GG36 protease, wherein said Bacillus lentussubtilisin GG36 protease comprises the amino acid sequence shown in SEQID NO:2, and wherein said subtilisin variant has at least 80% amino acidsequence identity to SEQ ID NO:2 and has proteolytic activity andcomprises an amino acid sequence comprising a combination of amino acidsubstitutions selected from List 6, and wherein amino acid positions ofthe subtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention further provides an isolated subtilisin variant ofBacillus lentus subtilisin GG36 protease, wherein said Bacillus lentussubtilisin GG36 protease comprises the amino acid sequence shown in SEQID NO:2, and wherein said subtilisin variant has at least 80% amino acidsequence identity to SEQ ID NO:2 and has proteolytic activity andcomprises an amino acid sequence comprising a combination of amino acidsubstitutions selected from List 7, and wherein amino acid positions ofthe subtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention further provides an isolated subtilisin variant ofBacillus lentus subtilisin GG36 protease, wherein said Bacillus lentussubtilisin GG36 protease comprises the amino acid sequence shown in SEQID NO:2, and wherein said subtilisin variant has at least 80% amino acidsequence identity to SEQ ID NO:2 and has proteolytic activity andcomprises an amino acid sequence comprising a combination of amino acidsubstitutions selected from List 8, and wherein amino acid positions ofthe subtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention further provides an isolated subtilisin variant ofBacillus lentus subtilisin GG36 protease, wherein said Bacillus lentussubtilisin GG36 protease comprises the amino acid sequence shown in SEQID NO:2, and wherein said subtilisin variant has at least 80% amino acidsequence identity to SEQ ID NO:2 and has proteolytic activity andcomprises an amino acid sequence comprising a combination of amino acidsubstitutions selected from List 9, and wherein amino acid positions ofthe subtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention further provides an isolated subtilisin variant ofBacillus lentus subtilisin GG36 protease, wherein said Bacillus lentussubtilisin GG36 protease comprises the amino acid sequence shown in SEQID NO:2, and wherein said subtilisin variant has at least 80% amino acidsequence identity to SEQ ID NO:2 and has proteolytic activity andcomprises an amino acid sequence comprising a combination of amino acidsubstitutions selected from List 10, and wherein amino acid positions ofthe subtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention further provides an isolated subtilisin variant ofBacillus lentus subtilisin GG36 protease, wherein said Bacillus lentussubtilisin GG36 protease comprises the amino acid sequence shown in SEQID NO:2, and wherein said subtilisin variant has at least 80% amino acidsequence identity to SEQ ID NO:2 and has proteolytic activity andcomprises an amino acid sequence comprising a combination of amino acidsubstitutions selected from List 11, and wherein amino acid positions ofthe subtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention further provides an isolated subtilisin variant ofBacillus lentus subtilisin GG36 protease, wherein said Bacillus lentussubtilisin GG36 protease comprises the amino acid sequence shown in SEQID NO:2, and wherein said subtilisin variant has at least 80% amino acidsequence identity to SEQ ID NO:2 and has proteolytic activity andcomprises an amino acid sequence comprising a combination of amino acidsubstitutions selected from List 12, and wherein amino acid positions ofthe subtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention further provides an isolated subtilisin variant ofBacillus lentus subtilisin GG36 protease, wherein said Bacillus lentussubtilisin GG36 protease comprises the amino acid sequence shown in SEQID NO:2, and wherein said subtilisin variant has at least 80% amino acidsequence identity to SEQ ID NO:2 and has proteolytic activity andcomprises an amino acid sequence comprising a combination of amino acidsubstitutions selected from List 13, and wherein amino acid positions ofthe subtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention further provides an isolated subtilisin variant ofBacillus lentus subtilisin GG36 protease, wherein said Bacillus lentussubtilisin GG36 protease comprises the amino acid sequence shown in SEQID NO:2, and wherein said subtilisin variant has at least 80% amino acidsequence identity to SEQ ID NO:2 and has proteolytic activity andcomprises an amino acid sequence comprising a combination of amino acidsubstitutions selected from List 14, and wherein amino acid positions ofthe subtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention further provides an isolated subtilisin variant ofBacillus lentus subtilisin GG36 protease, wherein said Bacillus lentussubtilisin GG36 protease comprises the amino acid sequence shown in SEQID NO:2, and wherein said subtilisin variant has at least 80% amino acidsequence identity to SEQ ID NO:2 and has proteolytic activity andcomprises an amino acid sequence comprising a combination of amino acidsubstitutions selected from List 15, and wherein amino acid positions ofthe subtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention further provides an isolated subtilisin variant ofBacillus lentus subtilisin GG36 protease, wherein said Bacillus lentussubtilisin GG36 protease comprises the amino acid sequence shown in SEQID NO:2, and wherein said subtilisin variant has at least 80% amino acidsequence identity to SEQ ID NO:2 and has proteolytic activity andcomprises an amino acid sequence comprising a combination of amino acidsubstitutions selected from List 16, and wherein amino acid positions ofthe subtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention further provides an isolated subtilisin variant ofBacillus lentus subtilisin GG36 protease, wherein said Bacillus lentussubtilisin GG36 protease comprises the amino acid sequence shown in SEQID NO:2, and wherein said subtilisin variant has at least 80% amino acidsequence identity to SEQ ID NO:2 and has proteolytic activity andcomprises an amino acid sequence comprising a combination of amino acidsubstitutions selected from List 17, and wherein amino acid positions ofthe subtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention further provides an isolated subtilisin variant ofBacillus lentus subtilisin GG36 protease, wherein said Bacillus lentussubtilisin GG36 protease comprises the amino acid sequence shown in SEQID NO:2, and wherein said subtilisin variant has at least 80% amino acidsequence identity to SEQ ID NO:2 and has proteolytic activity andcomprises an amino acid sequence comprising a combination of amino acidsubstitutions selected from List 18, and wherein amino acid positions ofthe subtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention further provides an isolated subtilisin variant ofBacillus lentus subtilisin GG36 protease, wherein said Bacillus lentussubtilisin GG36 protease comprises the amino acid sequence shown in SEQID NO:2, and wherein said subtilisin variant has at least 80% amino acidsequence identity to SEQ ID NO:2 and has proteolytic activity andcomprises an amino acid sequence comprising a combination of amino acidsubstitutions selected from List 19, and wherein amino acid positions ofthe subtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention further provides an isolated subtilisin variant ofBacillus lentus subtilisin GG36 protease, as listed above, wherein saidsubtilisin variant has at least 85% amino acid identity with saidBacillus lentus subtilisin GG36 protease comprising the amino acidsequence shown in SEQ ID NO:2.

The present invention further provides an isolated subtilisin variant ofBacillus lentus subtilisin GG36 protease, as listed above, wherein saidsubtilisin variant has at least 90% amino acid identity with saidBacillus lentus subtilisin GG36 protease comprising the amino acidsequence shown in SEQ ID NO:2.

The present invention further provides an isolated subtilisin variant ofBacillus lentus subtilisin GG36 protease, as listed above, wherein saidsubtilisin variant has at least 95% amino acid identity with saidBacillus lentus subtilisin GG36 protease comprising the amino acidsequence shown in SEQ ID NO:2.

The present invention further provides an isolated subtilisin variant ofBacillus lentus subtilisin GG36 protease, as listed above, wherein saidsubtilisin variant has at least 98% amino acid identity with saidBacillus lentus subtilisin GG36 protease comprising the amino acidsequence shown in SEQ ID NO:2.

The present invention further provides an isolated subtilisin variant ofBacillus lentus subtilisin GG36 protease, as listed above, wherein saidsubtilisin variant has at least 99% amino acid identity with saidBacillus lentus subtilisin GG36 protease comprising the amino acidsequence shown in SEQ ID NO:2.

The present invention further provides any of the isolated subtilisinvariants listed above, wherein the total net charge of the variant is 0,+1, +2, +3, +4, +5, −1, −2, −3, −4, or −5 relative to the total netcharge of the Bacillus lentus subtilisin GG36 protease. In someembodiments, the total net charge is obtained by one or moresubstitutions selected from: A001E, V004E, R010H/A, Q012E, A015D, N018D,R019H/S, V026D, K027E, N043D, R045C/T/S/P, S049D, V051E, G061E, N062D/E,N076D, N077D, S078D, S087D, A098E, S101D, S106E, G115E, G118D, N123D,S128D, P129E, S130D/E, S132D, A158E, G159D/E, S160D, S166D, R170T,N183D, N184D, N185E, R186H, S188D/E, A194E, A200D, Y209E, A215D, N204D,S212D, L217D/E, N218D, A230E, K235F, N237D, K237E, N238D, S240D, N243D,Q245D, R247L, N248D/E, K251C, N263D, N269D/E, A272D, A273E, R275H/S,A001R, V004R, Q012R, P014R, H017R, N018R/K, G020K/R, T022R/K, S024R/K,G025R, D032I, D041K/L/N, N043R/K, G046R, A048R, F050R/K, P055R, S056R/K,T057R, Q059K, G061R, N076K, S078R, P086R, S087R, E089G/P/I, G097R,S099R, Q109R, G115R, G118R, S132K, S144R, G159R/K, D181C/S/T, L196K,N204K, Q206R, K235R, Q236R/K, X237R, N238R, S240R, W241R, S242R/K,V244R, Q245R/K, N248R, H249R, N252R/K, S256R, G258R, T260K, N269R/K,and/or E271F/H/T/L/W/R/S/I/A/G/V, and wherein amino acid positions ofthe protease variant, more specifically subtilisin variants are numberedaccording to the numbering of corresponding amino acid positions in theamino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ shownin SEQ ID NO:1.

The present invention also provides isolated protease variants, morespecifically subtilisin variants, wherein said protease variant, morespecifically subtilisin variants has at least one, or even two or morecharged mutations selected from the group consisting of A001E, V004E,R010H/A, Q012E, A015D, N018D, R019H/S, V026D, K027E, N043D, R045C/T/S/P,S049D, V051E, G061E, N062D/E, N076D, N077D, S078D, S087D, A098E, S101D,S106E, G115E, G118D, N123D, S128D, P129E, S130D/E, S132D, A158E,G159D/E, S160D, S166D, R170T, N183D, N184D, N185E, R186H, S188D/E,A194E, A200D, Y209E, A215D, N204D, S212D, L217D/E, N218D, A230E, K235F,N237D, K237E, N238D, S240D; N243D, Q245D, R247L, N248D/E, K251C, N263D,N269D/E, A272D, A273E, and/or R275H/S, preferably having a charge of 0,−1, −2, −3, −4 or −5, preferably 0, −1, −2 or −3, most preferably −1 or−2 relative to the enzyme of SEQ ID NO:1. Said protease variants, morespecifically subtilisin variants can also include any of the variantslisted in the application.

The present invention also provides isolated protease variants, morespecifically subtilisin variants, wherein said protease variant, morespecifically subtilisin variants has one or two or more chargedmutations selected from the group consisting of A001R, V004R, Q012R,P014R, H017R, N018R/K, G020K/R, T022R/K, S024R/K, G025R, D032I,D041K/L/N, N043R/K, G046R, A048R, F050R/K, P055R, S056R/K, T057R, Q059K,G061R, N076K, S078R, P086R, S087R, E089G/P/I, G097R, S099R, Q109R,G115R, G118R, S132K, S144R, G159R/K, D181C/S/T, L196K, N204K, Q206R,K235R, Q236R/K, K237R, N238R, S240R, W241R, S242R/K, V244R, Q245R/K,N248R, H249R, N252R/K, S256R, G258R, T260K, N269R/K, and/orE271F/H/T/L/W/R/S/I/A/V, preferably having a charge of 0, +1, +2, +3, +4or +5, preferably +1, +2 or +3, most preferably +2 relative to theenzyme of SEQ ID NO:1. Said protease variants, more specificallysubtilisin variants can also include any of the variants listed in theapplication.

In some embodiments, any of the above listed isolated subtilisinvariants can be incorporated into a detergent composition suitable foraddition to water to make a wash liquor having low ionic strength or lowdetergent concentration. Thus in a preferred aspect of the invention,these variants will form part of a detergent composition that is addedto water, either for a handwashing or machine washing process, typicallywithin a washing machine, to form a wash liquor, whose conductivity isfrom about 0.1 mS/cm to about 3 mS/cm, from about 0.3 mS/cm to about 2.5mS/cm, or even from about 0.5 mS/cm to about 2 mS/cm. Preferred variantsfor use in low ionic strength or low detergent concentration areselected from the variants in any of lists 9, 14, 15 or 16, preferablylist 14 or 15, most preferably list 15 above.

Without wishing to be bound by theory it is believed that thesemutations to arrive at a desired net charge provide enhanced overallprotease performance by ensuring optimal charge of the molecule for lowionic strength conditions, or wash liquors comprising low detergentconcentration—it is only through careful combination of certainmutations, of which these are preferred, that such preferred proteasescan be obtained.

In some embodiments, any of the above listed isolated subtilisinvariants can be incorporated into a detergent composition suitable foraddition to water to make a wash liquor having high ionic strength orhigh detergent concentration. Thus in a preferred aspect of theinvention, these variants will form part of a detergent composition thatis added to water, either for a handwashing or machine washing process,typically within a washing machine, to form a wash liquor, whoseconductivity is from above about 3 mS/cm to about 30 mS/cm, from about3.5 mS/cm to about 20 mS/cm, or even from about 4 mS/cm to about 10mS/cm. Preferred variants for use in high ionic strength or highdetergent concentration are selected from the variants in any of lists 1or 17, 18 or 19, preferably list 17 or 18, most preferably list 17above.

Without wishing to be bound by theory it is believed that thesemutations to arrive at a desired net charge provide enhanced overallprotease performance by ensuring optimal charge of the molecule for highionic strength conditions, or wash liquors comprising high detergentconcentration—it is only through careful combination of certainmutations, of which these are preferred, that such preferred proteasescan be obtained.

Preferably these proteases form part of a detergent composition that isadded to water either in a hand or machine washing process, typicallywithin a washing machine, to form a wash liquor, whose conductivity isfrom above about 3 mS/cm to about 30 mS/cm, from about 3.5 mS/cm toabout 20 mS/cm, or even from about 4 mS/cm to about 10 mS/cm.

Without wishing to be bound by theory it is believed that thesemutations to arrive at a desired net charge provide enhanced overallprotease performance in high ionic strength or high detergentconcentration conditions—it is only through careful combination ofcertain mutations, of which these are preferred, that such preferredproteases can be obtained.

The present invention further provides any of the isolated subtilisinvariants listed above having one or more of the followingcharacteristics: a) a Test Method 2 performance index of at least 1.1,at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, atleast 1.7, at least 1.8, at least 1.9, at least 2, from 1.1 to about 10,from 1.1 to about 8, or even from 1.1 to about 5; b) a Test Method 3performance index of at least 1.1, at least 1.2, at least 1.3, at least1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least1.9, at least 2, from 1.1 to about 10, from 1.1 to about 8, or even from1.1 to about 5; c) a Test Method 4 performance index of at least 1.0, atleast 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, atleast 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from1.0 to about 10, from 1.0 to about 8, or even from 1.0 to about 5;and/or d) a Test Method 6 performance index of at least 1.0, at least1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from 1.0 toabout 10, from 1.0 to about 8, or even from 1.0 to about 5; and/or e)Test Method 7 performance index of at least 1.1, at least 1.2, at least1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least1.8, at least 1.9, at least 2, from 1.1 to about 15, from 1.1 to about10, or even from 1.1 to about 7. Test Method 2, Test Method 3, TestMethod 4, Test Method 6, and Test Method 7 are explicitly describedinfra in the section of Example 1 entitled “Test Methods”.

Nucleic Acids of the Invention

The invention provides isolated, non-naturally occurring, or recombinantnucleic acids (also referred to herein as “polynucleotides”), which maybe collectively referred to as “nucleic acids of the invention” or“polynucleotides of the invention”, which encode polypeptides of theinvention. Nucleic acids of the invention, including all describedbelow, are useful in recombinant production (e.g., expression) ofpolypeptides of the invention, typically through expression of a plasmidexpression vector comprising a sequence encoding the polypeptide ofinterest or fragment thereof. As discussed above, polypeptides includevariant protease polypeptides, including variant subtilisin polypeptideshaving enzymatic activity (e.g., proteolytic activity) which are usefulin cleaning applications and cleaning compositions for cleaning an itemor a surface (e.g., surface of an item) in need of cleaning.

In some embodiments, the invention provides an isolated, recombinant,substantially pure, or non-naturally occurring nucleic acid comprising anucleotide sequence encoding any polypeptide (including any fusionprotein, etc.) of the invention described above in the section entitled“Polypeptides of the Invention” and elsewhere herein. The invention alsoprovides an isolated, recombinant, substantially pure, ornon-naturally-occurring nucleic acid comprising a nucleotide sequenceencoding a combination of two or more of any polypeptides of theinvention described above and elsewhere herein.

Also provided is an isolated, recombinant, substantially pure, ornon-naturally occurring nucleic acid comprising a polynucleotidesequence which encodes a variant protease having proteolytic activity,said variant protease (e.g., variant subtilisin) comprising an aminoacid sequence which differs from the amino acid sequence of SEQ ID NO:2by no more than 50, no more than 40, no more than 30, no more than 35,no more than 25, no more than 20, no more than 19, no more than 18, nomore than 17, no more than 16, no more than 15, no more than 14, no morethan 13, no more than 12, no more than 11, no more than 10, no more than9, no more than 8, no more than 7, no more than 6, no more than 5, nomore than 4, no more than 3, no more than 2, or no more than 1 aminoacid residue(s), wherein amino acid positions of the variant subtilisinare numbered according to the numbering of corresponding amino acidpositions in the amino acid sequence of Bacillus amyloliquefacienssubtilisin BPN′ shown in SEQ ID NO:1 as determined by alignment of thevariant protease amino acid sequence with the Bacillus amyloliquefacienssubtilisin BPN′ amino acid sequence.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant, wherein said subtilisin variant is a mature formhaving proteolytic activity and comprises an amino acid sequencecomprising a combination of amino acid substitutions at positionsselected from List 1, and wherein amino acid positions of the subtilisinvariant are numbered according to the numbering of corresponding aminoacid positions in the amino acid sequence of Bacillus amyloliquefacienssubtilisin BPN′ shown in SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant, wherein said subtilisin variant is a mature formhaving proteolytic activity and comprises an amino acid sequencecomprising a combination of amino acid substitutions at positionsselected from List 2, and wherein amino acid positions of the subtilisinvariant are numbered according to the numbering of corresponding aminoacid positions in the amino acid sequence of Bacillus amyloliquefacienssubtilisin BPN′ shown in SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant, wherein said subtilisin variant is a mature formhaving proteolytic activity and comprises an amino acid sequencecomprising a combination of amino acid substitutions at positionsselected from List 3, and wherein amino acid positions of the subtilisinvariant are numbered according to the numbering of corresponding aminoacid positions in the amino acid sequence of Bacillus amyloliquefacienssubtilisin BPN′ shown in SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant, wherein said subtilisin variant is a mature formhaving proteolytic activity and comprises an amino acid sequencecomprising a combination of amino acid substitutions at positionsselected from List 4, and wherein amino acid positions of the subtilisinvariant are numbered according to the numbering of corresponding aminoacid positions in the amino acid sequence of Bacillus amyloliquefacienssubtilisin BPN′ shown in SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant, wherein said subtilisin variant is a mature formhaving proteolytic activity and comprises an amino acid sequencecomprising a combination of amino acid substitutions at positionsselected from List 5, and wherein amino acid positions of the subtilisinvariant are numbered according to the numbering of corresponding aminoacid positions in the amino acid sequence of Bacillus amyloliquefacienssubtilisin BPN′ shown in SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant, wherein said subtilisin variant is a mature formhaving proteolytic activity and comprises an amino acid sequencecomprising a combination of amino acid substitutions at positionsselected from List 6, and wherein amino acid positions of the subtilisinvariant are numbered according to the numbering of corresponding aminoacid positions in the amino acid sequence of Bacillus amyloliquefacienssubtilisin BPN′ shown in SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant, wherein said subtilisin variant is a mature formhaving proteolytic activity and comprises an amino acid sequencecomprising a combination of amino acid substitutions at positionsselected from List 7, and wherein amino acid positions of the subtilisinvariant are numbered according to the numbering of corresponding aminoacid positions in the amino acid sequence of Bacillus amyloliquefacienssubtilisin BPN′ shown in SEQ ID NO:1.

The present invention provides an isolated subtilisin variant, whereinsaid subtilisin variant is a mature form having proteolytic activity andcomprises an amino acid sequence comprising a combination of amino acidsubstitutions at positions selected from List 8, and wherein amino acidpositions of the subtilisin variant are numbered according to thenumbering of corresponding amino acid positions in the amino acidsequence of Bacillus amyloliquefaciens subtilisin BPN′ shown in SEQ IDNO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant, wherein said subtilisin variant is a mature formhaving proteolytic activity and comprises an amino acid sequencecomprising a combination of amino acid substitutions at positionsselected from List 9, and wherein amino acid positions of the subtilisinvariant are numbered according to the numbering of corresponding aminoacid positions in the amino acid sequence of Bacillus amyloliquefacienssubtilisin BPN′ shown in SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant, wherein said subtilisin variant is a mature formhaving proteolytic activity and comprises an amino acid sequencecomprising a combination of amino acid substitutions at positionsselected from List 10, and wherein amino acid positions of thesubtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant, wherein said subtilisin variant is a mature formhaving proteolytic activity and comprises an amino acid sequencecomprising a combination of amino acid substitutions at positionsselected from List 11, and wherein amino acid positions of thesubtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant, wherein said subtilisin variant is a mature formhaving proteolytic activity and comprises an amino acid sequencecomprising a combination of amino acid substitutions at positionsselected from List 12, and wherein amino acid positions of thesubtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention provides an isolated subtilisin variant, whereinsaid subtilisin variant is a mature form having proteolytic activity andcomprises an amino acid sequence comprising a combination of amino acidsubstitutions at positions selected from List 13, and wherein amino acidpositions of the subtilisin variant are numbered according to thenumbering of corresponding amino acid positions in the amino acidsequence of Bacillus amyloliquefaciens subtilisin BPN′ shown in SEQ IDNO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant, wherein said subtilisin variant is a mature formhaving proteolytic activity and comprises an amino acid sequencecomprising a combination of amino acid substitutions at positionsselected from List 14, and wherein amino acid positions of thesubtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant, wherein said subtilisin variant is a mature formhaving proteolytic activity and comprises an amino acid sequencecomprising a combination of amino acid substitutions at positionsselected from List 15, and wherein amino acid positions of thesubtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant, wherein said subtilisin variant is a mature formhaving proteolytic activity and comprises an amino acid sequencecomprising a combination of amino acid substitutions at positionsselected from List 16, and wherein amino acid positions of thesubtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant, wherein said subtilisin variant is a mature formhaving proteolytic activity and comprises an amino acid sequencecomprising a combination of amino acid substitutions at positionsselected from List 17, and wherein amino acid positions of thesubtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention provides an isolated subtilisin variant, whereinsaid subtilisin variant is a mature form having proteolytic activity andcomprises an amino acid sequence comprising a combination of amino acidsubstitutions at positions selected from List 18, and wherein amino acidpositions of the subtilisin variant are numbered according to thenumbering of corresponding amino acid positions in the amino acidsequence of Bacillus amyloliquefaciens subtilisin BPN′ shown in SEQ IDNO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant, wherein said subtilisin variant is a mature formhaving proteolytic activity and comprises an amino acid sequencecomprising a combination of amino acid substitutions at positionsselected from List 19, and wherein amino acid positions of thesubtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention provides nucleic acids encoding any of the aboveisolated subtilisin variants, wherein the subtilisin variant is aprotease variant of Bacillus lentus subtilisin GG36 protease, whereinsaid Bacillus lentus subtilisin GG36 protease comprises the amino acidsequence shown in SEQ ID NO:2.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant of Bacillus lentus subtilisin GG36 protease, whereinsaid Bacillus lentus subtilisin GG36 protease comprises the amino acidsequence shown in SEQ ID NO:2, and wherein said subtilisin variant hasat least 80% amino acid sequence identity to SEQ ID NO:2 and hasproteolytic activity and comprises an amino acid sequence comprising acombination of amino acid substitutions selected from List 1, andwherein amino acid positions of the subtilisin variant are numberedaccording to the numbering of corresponding amino acid positions in theamino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ shownin SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant of Bacillus lentus subtilisin GG36 protease, whereinsaid Bacillus lentus subtilisin GG36 protease comprises the amino acidsequence shown in SEQ ID NO:2, and wherein said subtilisin variant hasat least 80% amino acid sequence identity to SEQ ID NO:2 and hasproteolytic activity and comprises an amino acid sequence comprising acombination of amino acid substitutions selected from List 2, andwherein amino acid positions of the subtilisin variant are numberedaccording to the numbering of corresponding amino acid positions in theamino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ shownin SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant of Bacillus lentus subtilisin GG36 protease, whereinsaid Bacillus lentus subtilisin GG36 protease comprises the amino acidsequence shown in SEQ ID NO:2, and wherein said subtilisin variant hasat least 80% amino acid sequence identity to SEQ ID NO:2 and hasproteolytic activity and comprises an amino acid sequence comprising acombination of amino acid substitutions selected from List 3, andwherein amino acid positions of the subtilisin variant are numberedaccording to the numbering of corresponding amino acid positions in theamino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ shownin SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant of Bacillus lentus subtilisin GG36 protease, whereinsaid Bacillus lentus subtilisin GG36 protease comprises the amino acidsequence shown in SEQ ID NO:2, and wherein said subtilisin variant hasat least 80% amino acid sequence identity to SEQ ID NO:2 and hasproteolytic activity and comprises an amino acid sequence comprising acombination of amino acid substitutions selected from List 4, andwherein amino acid positions of the subtilisin variant are numberedaccording to the numbering of corresponding amino acid positions in theamino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ shownin SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant of Bacillus lentus subtilisin GG36 protease, whereinsaid Bacillus lentus subtilisin GG36 protease comprises the amino acidsequence shown in SEQ ID NO:2, and wherein said subtilisin variant hasat least 80% amino acid sequence identity to SEQ ID NO:2 and hasproteolytic activity and comprises an amino acid sequence comprising acombination of amino acid substitutions selected from List 5, andwherein amino acid positions of the subtilisin variant are numberedaccording to the numbering of corresponding amino acid positions in theamino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ shownin SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant of Bacillus lentus subtilisin GG36 protease, whereinsaid Bacillus lentus subtilisin GG36 protease comprises the amino acidsequence shown in SEQ ID NO:2, and wherein said subtilisin variant hasat least 80% amino acid sequence identity to SEQ ID NO:2 and hasproteolytic activity and comprises an amino acid sequence comprising acombination of amino acid substitutions selected from List 6, andwherein amino acid positions of the subtilisin variant are numberedaccording to the numbering of corresponding amino acid positions in theamino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ shownin SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant of Bacillus lentus subtilisin GG36 protease, whereinsaid Bacillus lentus subtilisin GG36 protease comprises the amino acidsequence shown in SEQ ID NO:2, and wherein said subtilisin variant hasat least 80% amino acid sequence identity to SEQ ID NO:2 and hasproteolytic activity and comprises an amino acid sequence comprising acombination of amino acid substitutions selected from List 7, andwherein amino acid positions of the subtilisin variant are numberedaccording to the numbering of corresponding amino acid positions in theamino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ shownin SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant of Bacillus lentus subtilisin GG36 protease, whereinsaid Bacillus lentus subtilisin GG36 protease comprises the amino acidsequence shown in SEQ ID NO:2, and wherein said subtilisin variant hasat least 80% amino acid sequence identity to SEQ ID NO:2 and hasproteolytic activity and comprises an amino acid sequence comprising acombination of amino acid substitutions selected from List 8, andwherein amino acid positions of the subtilisin variant are numberedaccording to the numbering of corresponding amino acid positions in theamino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ shownin SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant of Bacillus lentus subtilisin GG36 protease, whereinsaid Bacillus lentus subtilisin GG36 protease comprises the amino acidsequence shown in SEQ ID NO:2, and wherein said subtilisin variant hasat least 80% amino acid sequence identity to SEQ ID NO:2 and hasproteolytic activity and comprises an amino acid sequence comprising acombination of amino acid substitutions selected from List 9, andwherein amino acid positions of the subtilisin variant are numberedaccording to the numbering of corresponding amino acid positions in theamino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ shownin SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant of Bacillus lentus subtilisin GG36 protease, whereinsaid Bacillus lentus subtilisin GG36 protease comprises the amino acidsequence shown in SEQ ID NO:2, and wherein said subtilisin variant hasat least 80% amino acid sequence identity to SEQ ID NO:2 and hasproteolytic activity and comprises an amino acid sequence comprising acombination of amino acid substitutions selected from List 10, andwherein amino acid positions of the subtilisin variant are numberedaccording to the numbering of corresponding amino acid positions in theamino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ shownin SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant of Bacillus lentus subtilisin GG36 protease, whereinsaid Bacillus lentus subtilisin GG36 protease comprises the amino acidsequence shown in SEQ ID NO:2, and wherein said subtilisin variant hasat least 80% amino acid sequence identity to SEQ ID NO:2 and hasproteolytic activity and comprises an amino acid sequence comprising acombination of amino acid substitutions selected from List 11, andwherein amino acid positions of the subtilisin variant are numberedaccording to the numbering of corresponding amino acid positions in theamino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ shownin SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant of Bacillus lentus subtilisin GG36 protease, whereinsaid Bacillus lentus subtilisin GG36 protease comprises the amino acidsequence shown in SEQ ID NO:2, and wherein said subtilisin variant hasat least 80% amino acid sequence identity to SEQ ID NO:2 and hasproteolytic activity and comprises an amino acid sequence comprising acombination of amino acid substitutions selected from List 12, andwherein amino acid positions of the subtilisin variant are numberedaccording to the numbering of corresponding amino acid positions in theamino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ shownin SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant of Bacillus lentus subtilisin GG36 protease, whereinsaid Bacillus lentus subtilisin GG36 protease comprises the amino acidsequence shown in SEQ ID NO:2, and wherein said subtilisin variant hasat least 80% amino acid sequence identity to SEQ ID NO:2 and hasproteolytic activity and comprises an amino acid sequence comprising acombination of amino acid substitutions selected from List 13, andwherein amino acid positions of the subtilisin variant are numberedaccording to the numbering of corresponding amino acid positions in theamino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ shownin SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant of Bacillus lentus subtilisin GG36 protease, whereinsaid Bacillus lentus subtilisin GG36 protease comprises the amino acidsequence shown in SEQ ID NO:2, and wherein said subtilisin variant hasat least 80% amino acid sequence identity to SEQ ID NO:2 and hasproteolytic activity and comprises an amino acid sequence comprising acombination of amino acid substitutions selected from List 14, andwherein amino acid positions of the subtilisin variant are numberedaccording to the numbering of corresponding amino acid positions in theamino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ shownin SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant of Bacillus lentus subtilisin GG36 protease, whereinsaid Bacillus lentus subtilisin GG36 protease comprises the amino acidsequence shown in SEQ ID NO:2, and wherein said subtilisin variant hasat least 80% amino acid sequence identity to SEQ ID NO:2 and hasproteolytic activity and comprises an amino acid sequence comprising acombination of amino acid substitutions selected from List 15, andwherein amino acid positions of the subtilisin variant are numberedaccording to the numbering of corresponding amino acid positions in theamino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ shownin SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant of Bacillus lentus subtilisin GG36 protease, whereinsaid Bacillus lentus subtilisin GG36 protease comprises the amino acidsequence shown in SEQ ID NO:2, and wherein said subtilisin variant hasat least 80% amino acid sequence identity to SEQ ID NO:2 and hasproteolytic activity and comprises an amino acid sequence comprising acombination of amino acid substitutions selected from List 16, andwherein amino acid positions of the subtilisin variant are numberedaccording to the numbering of corresponding amino acid positions in theamino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ shownin SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant of Bacillus lentus subtilisin GG36 protease, whereinsaid Bacillus lentus subtilisin GG36 protease comprises the amino acidsequence shown in SEQ ID NO:2, and wherein said subtilisin variant hasat least 80% amino acid sequence identity to SEQ ID NO:2 and hasproteolytic activity and comprises an amino acid sequence comprising acombination of amino acid substitutions selected from List 17, andwherein amino acid positions of the subtilisin variant are numberedaccording to the numbering of corresponding amino acid positions in theamino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ shownin SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant of Bacillus lentus subtilisin GG36 protease, whereinsaid Bacillus lentus subtilisin GG36 protease comprises the amino acidsequence shown in SEQ ID NO:2, and wherein said subtilisin variant hasat least 80% amino acid sequence identity to SEQ ID NO:2 and hasproteolytic activity and comprises an amino acid sequence comprising acombination of amino acid substitutions selected from List 18, andwherein amino acid positions of the subtilisin variant are numberedaccording to the numbering of corresponding amino acid positions in theamino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ shownin SEQ ID NO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant of Bacillus lentus subtilisin GG36 protease, whereinsaid Bacillus lentus subtilisin GG36 protease comprises the amino acidsequence shown in SEQ ID NO:2, and wherein said subtilisin variant hasat least 80% amino acid sequence identity to SEQ ID NO:2 and hasproteolytic activity and comprises an amino acid sequence comprising acombination of amino acid substitutions selected from 19, and whereinamino acid positions of the subtilisin variant are numbered according tothe numbering of corresponding amino acid positions in the amino acidsequence of Bacillus amyloliquefaciens subtilisin BPN′ shown in SEQ IDNO:1.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant of Bacillus lentus subtilisin GG36 protease, aslisted above, wherein said subtilisin variant has at least 85% aminoacid identity with said Bacillus lentus subtilisin GG36 proteasecomprising the amino acid sequence shown in SEQ ID NO:2.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant of Bacillus lentus subtilisin GG36 protease, aslisted above, wherein said subtilisin variant has at least 90% aminoacid identity with said Bacillus lentus subtilisin GG36 proteasecomprising the amino acid sequence shown in SEQ ID NO:2.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant of Bacillus lentus subtilisin GG36 protease, aslisted above, wherein said subtilisin variant has at least 95% aminoacid identity with said Bacillus lentus subtilisin GG36 proteasecomprising the amino acid sequence shown in SEQ ID NO:2.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant of Bacillus lentus subtilisin GG36 protease, aslisted above, wherein said subtilisin variant has at least 98% aminoacid identity with said Bacillus lentus subtilisin GG36 proteasecomprising the amino acid sequence shown in SEQ ID NO:2.

The present invention provides nucleic acids encoding an isolatedsubtilisin variant of Bacillus lentus subtilisin GG36 protease, aslisted above, wherein said subtilisin variant has at least 99% aminoacid identity with said Bacillus lentus subtilisin GG36 proteasecomprising the amino acid sequence shown in SEQ ID NO:2.

The present invention further provides nucleic acids encoding any of theisolated subtilisin variants listed above, wherein the total net chargeof the variant is 0, +1, +2, +3, +4, +5, −1, −2, −3, −4, or −5 relativeto the total net charge of the Bacillus lentus subtilisin GG36 protease.In some embodiments, the total net charge is obtained by one or moresubstitutions selected from: A001E, V004E, R010H/A, Q012E, A015D, N018D,R019H/S, V026D, K027E, N043D, R045C/T/S/P, S049D, V051E, G061E, N062D/E,N076D, N077D, S078D, S087D, A098E, S101D, S106E, G115E, G118D, N123D,S128D, P129E, S130D/E, S132D, A158E, G159D/E, S160D, S166D, R170T,N183D, N184D, N185E, R186H, S188D/E, A194E, A200D, Y209E, A215D, N204D,S212D, L217D/E, N218D, A230E, K235F, N237D, K237E, N238D, S240D, N243D,Q245D, R247L, N248D/E, K251C, N263D, N269D/E, A272D, A273E, R275H/S,A001R, V004R, Q012R, P014R, H017R, N018R/K, G020K/R, T022R/K, S024R/K,G025R, D032I, D041K/L/N, N043R/K, G046R, A048R, F050R/K, P055R, S056R/K,T057R, Q059K, G061R, N076K, S078R, P086R, S087R, E089G/P/I, G097R,S099R, Q109R, G115R, G118R, S132K, S144R, G159R/K, D181C/S/T, L196K,N204K, Q206R, K235R, Q236R/K, K237R, N238R, S240R, W241R, S242R/K,V244R, Q245R/K, N248R, H249R, N252R/K, S256R, G258R, T260K, N269R/K, orE271F/H/T/L/W/R/S/I/A/G/V, and wherein amino acid positions of theprotease variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

The present invention further provides nucleic acids encoding isolatedprotease variants, more specifically subtilisin variants, wherein saidprotease variant has at least one, or even two or more charged mutationsselected from the group consisting of A001E, V004E, R010H/A, Q012E,A015D, N018D, R019H/S, V026D, K027E, N043D, R045C/T/S/P, S049D, V051E,G061E, N062D/E, N076D, N077D, S078D, S087D, A098E, S101D, S106E, G115E,G118D, N123D, S128D, P129E, S130D/E, S132D, A158E, G159D/E, S160D,S166D, R170T, N183D, N184D, N185E, R186H, S188D/E, A194E, A200D, Y209E,A215D, N204D, S212D, L217D/E, N218D, A230E, K235F, N237D, K237E, N238D,S240D, N243D, Q245D, R247L, N248D/E, K251C, N263D, N269D/E, A272D,A273E, and/or R275H/S, preferably having a charge of 0, −1, −2, −3, −4or −5, preferably 0, −1, −2 or −3, most preferably −1 or −2 relative tothe enzyme of SEQ ID NO:1. Said protease variants, more specificallysubtilisin variants can also include any of the variants listed in theapplication.

The present invention also provides isolated protease variants, morespecifically subtilisin variants, wherein said protease variant has oneor two or more charged mutations selected from the group consisting ofA001R, V004R, Q012R, P014R, H017R, N018R/K, G020K/R, T022R/K, S024R/K,G025R, D032I, D041K/L/N, N043R/K, G046R, A048R, F050R/K, P055R, S056R/K,T057R, Q059K, G061R, N076K, S078R, P086R, S087R, E089G/P/I, G097R,S099R, Q109R, G115R, G118R, S132K, S144R, G159R/K, D181C/S/T, L196K,N204K, Q206R, K235R, Q236R/K, K237R, N238R, S240R, W241R, S242R/K,V244R, Q245R/K, N248R, H249R, N252R/K, S256R, G258R, T260K, N269R/K,and/or E271F/H/T/L/W/R/S/I/A/G/V and/or N269R, preferably having acharge of 0, +1, +2, +3, +4 or +5, preferably +1, +2 or +3, mostpreferably +2 relative to the enzyme of SEQ ID NO:1. Said proteasevariants, more specifically subtilisin variants can also include any ofthe variants listed in the application.

As indicated herein, suitable cold water protease variants, orsubtilisin variants, are variants of a parent protease, said parentprotease's sequence being at least 97%, at least 99% or 100% identicalto the amino acid sequence of SEQ ID NO:1, said protease variant havingone or more of the following characteristics:

a) Test Method 2 performance index of at least 1.1, at least 1.2, atleast 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, atleast 1.8, at least 1.9, at least 2, from 1.1 to about 10, from 1.1 toabout 8, or even from 1.1 to about 5; b) a Test Method 3 performanceindex of at least 1.1, at least 1.2, at least 1.3, at least 1.4, atleast 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, atleast 2, from 1.1 to about 10, from 1.1 to about 8, or even from 1.1 toabout 5; c) a Test Method 4 performance index of at least 1.0, at least1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from 1.0 toabout 10, from 1.0 to about 8, or even from 1.0 to about 5; and/or d) aTest Method 6 performance index of at least 1.0, at least 1.1, at least1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least1.7, at least 1.8, at least 1.9, at least 2, from 1.0 to about 10, from1.0 to about 8, or even from 1.0 to about 5; and/or e) Test Method 7performance index of at least 1.1, at least 1.2, at least 1.3, at least1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least1.9, at least 2, from 1.1 to about 15, from 1.1 to about 10, or evenfrom 1.1 to about 7. Test Method 2, Test Method 3, Test Method 4, TestMethod 6, and Test Method 7 are explicitly described infra in thesection of Example 1 entitled “Test Methods”. All mutations referencedherein utilize the BPN′ numbering scheme as shown in FIG. 1. In someembodiments, the variants referenced herein refer to variants havingamino acid sequences compared to the amino acid sequence of SEQ ID NO:2,using the BPN′ numbering scheme.

In some embodiments, the above high ionic strength cold water proteasevariants, more specifically subtilisin variants form part of a detergentcomposition that is diluted in water, typically within a laundry washingmachine, to form a laundry detergent wash liquor, whose conductivity isfrom about 3 mS/cm to about 30 mS/cm, from about 3.5 mS/cm to about 20mS/cm, or even from about 4 mS/cm to about 10 mS/cm.

The charge of the cold water protease variants, more specificallysubtilisin variants is expressed relative to B. lentus subtilisin GG36protease wild-type having the amino acid sequence of SEQ ID NO:2. Theamino acids that, impart a single negative charge are D and E and thosethat impart a single positive charge are R, H and K. Any amino acidchange versus SEQ ID NO:2 that changes a charge is used to calculate thecharge of the cold water protease variant. For example, introducing anegative charge mutation from a wild-type neutral position will add anet charge of −1 to the cold water protease variant, whereas introducinga negative charge mutation (D or E) from a wild-type positive amino acidresidue (R, H or K) will add a net charge of −2. Summing the chargechanges from all the amino acid residues that are different for the coldwater protease variant versus B. lentus subtilisin GG36 proteasewild-type having the amino acid sequence of SEQ ID NO:2 gives the chargechange of the cold water protease variant. Without wishing to be boundby theory, it is believed that: the preferred charge range for coldwater proteases to be used in low conductivity laundry detergentsolutions is −5, −4, −3, −2, −1, 0, particularly −2, −1; the preferredcharge range for cold water proteases to be used in high conductivitylaundry detergent solutions is +5, +4, +3, +2, +1, 0, particularly +2,+1. By correctly selecting the charge unexpectedly improved levels ofcold water cleaning performance can be obtained. “Low conductivitylaundry detergent solutions” are defined as having a conductivity offrom about 0.1 mS/cm to about 3 mS/cm, from about 0.3 mS/cm to about 2.5mS/cm, or even from about 0.5 mS/cm to about 2 mS/cm. “High conductivitylaundry detergent solutions” are defined as having a conductivity offrom about 3 mS/cm to about 30 mS/cm, from about 3.5 mS/cm to about 20mS/cm, or even from about 4 mS/cm to about 10 mS/cm. It is intended thatthe above examples be non-limiting. Once mutations are combined tooptimize cold water performance, the enzyme charge can also be balancedby mutations in further positions.

In some embodiments, the invention provides an isolated, recombinant,substantially pure, or non-naturally occurring variant protease (e.g.,variant subtilisin) having proteolytic activity, said variant proteasecomprising an amino acid sequence which differs from the amino acidsequence shown in SEQ ID NO:2 by no more than 50, no more than 45, nomore than 40, no more than 35, no more than 30, no more than 25, no morethan 20, no more than 19, no more than 18, no more than 17, no more than16, no more than 15, no more than 14, no more than 13, no more than 12,no more than 11, no more than 10, no more than 9, or no more than 8amino acid residues, wherein amino acid positions are numbered accordingto the numbering of corresponding amino acid positions in the amino acidsequence of Bacillus amyloliquefaciens subtilisin BPN′ shown in SEQ IDNO:1, as determined by alignment of the variant protease amino acidsequence with the Bacillus amyloliquefaciens subtilisin BPN′ amino acidsequence.

Nucleic acids of the invention can be generated by using any suitablesynthesis, manipulation, and/or isolation techniques, or combinationsthereof. For example, a polynucleotide of the invention may be producedusing standard nucleic acid synthesis techniques, such as solid-phasesynthesis techniques that are well-known to those skilled in the art. Insuch techniques, fragments of up to 50 or more nucleotide bases aretypically synthesized, then joined (e.g., by enzymatic or chemicalligation methods, or polymerase mediated recombination methods) to formessentially any desired continuous nucleic acid sequence. The synthesisof the nucleic acids of the invention can be also facilitated (oralternatively accomplished) by any suitable method known in the art,including but not limited to chemical synthesis using the classicalphosphoramidite method (See e.g., Beaucage et al. Tetrahedron Letters22:1859-69 [1981]); or the method described by Matthes et al. (See,Matthes et al., EMBO J. 3:801-805 [1984], as is typically practiced inautomated synthetic methods. Nucleic acids of the invention also can beproduced by using an automatic DNA synthesizer. Customized nucleic acidscan be ordered from a variety of commercial sources (e.g., The MidlandCertified Reagent Company, the Great American Gene Company, OperonTechnologies Inc., and DNA2.0). Other techniques for synthesizingnucleic acids and related principles are known in the art (See e.g.,Itakura et al., Ann. Rev. Biochem. 53:323 [1984]; and Itakura et al.,Science 198:1056 [1984]).

As indicated above, recombinant DNA techniques useful in modification ofnucleic acids are well known in the art. For example, techniques such asrestriction endonuclease digestion, ligation, reverse transcription andcDNA production, and polymerase chain reaction (e.g., PCR) are known andreadily employed by those of skill in the art. Nucleotides of theinvention may also be obtained by screening cDNA libraries (e.g., cDNAlibraries generated using mutagenesis techniques commonly used in theart, including those described herein) using one or more oligonucleotideprobes that can hybridize to or PCR-amplify polynucleotides which encodea variant protease polypeptide(s) of the invention. Procedures forscreening and isolating cDNA clones and PCR amplification procedures arewell known to those of skill in the art and described in standardreferences known to those skilled in the art. Some nucleic acids of theinvention can be obtained by altering a naturally occurringpolynucleotide backbone (e.g., that encodes an enzyme or parentprotease) by, for example, a known mutagenesis procedure (e.g.,site-directed mutagenesis, site saturation mutagenesis, and in vitrorecombination).

Methods for Making Modified Variant Proteases of the Invention

A variety of methods are known in the art that are suitable forgenerating modified polynucleotides of the invention that encode variantproteases of the invention, including, but not limited to, for example,site-saturation mutagenesis, scanning mutagenesis, insertionalmutagenesis, deletion mutagenesis, random mutagenesis, site-directedmutagenesis, and directed-evolution, as well as various otherrecombinatorial approaches. Methods for making modified polynucleotidesand proteins (e.g., variant proteases) include DNA shufflingmethodologies, methods based on non-homologous recombination of genes,such as ITCHY (See, Ostermeier et al., 7:2139-44 [1999]), SCRACHY (See,Lutz et al. 98:11248-53 [2001]), SHIPREC (See, Sieber et al., 19:456-60[2001]), and NRR (See, Bittker et al., 20:1024-9 [2001]; Bittker et al.,101:7011-6 [2004]), and methods that rely on the use of oligonucleotidesto insert random and targeted mutations, deletions and/or insertions(See, Ness et al., 20:1251-5 [2002]; Coco et al., 20:1246-50 [2002]; Zhaet al., 4:34-9 [2003]; Glaser et al., 149:3903-13 [1992]).

Vectors, Cells, and Methods for Producing Variant Proteases of theInvention

The present invention provides isolated or recombinant vectorscomprising at least one polynucleotide of the invention described herein(e.g., a polynucleotide encoding a variant protease of the inventiondescribed herein), isolated or recombinant expression vectors orexpression cassettes comprising at least one nucleic acid orpolynucleotide of the invention, isolated, substantially pure, orrecombinant DNA constructs comprising at least one nucleic acid orpolynucleotide of the invention, isolated or recombinant cellscomprising at least one polynucleotide of the invention, cell culturescomprising cells comprising at least one polynucleotide of theinvention, cell cultures comprising at least one nucleic acid orpolynucleotide of the invention, and compositions comprising one or moresuch vectors, nucleic acids, expression vectors, expression cassettes,DNA constructs, cells, cell cultures, or any combination or mixturesthereof.

In some embodiments, the invention provides recombinant cells comprisingat least one vector (e.g., expression vector or DNA construct) of theinvention which comprises at least one nucleic acid or polynucleotide ofthe invention. Some such recombinant cells are transformed ortransfected with such at least one vector. Such cells are typicallyreferred to as host cells. Some such cells comprise bacterial cells,including, but are not limited to Bacillus sp. cells, such as B.subtilis cells. The invention also provides recombinant cells (e.g.,recombinant host cells) comprising at least one variant protease of theinvention.

In some embodiments, the invention provides a vector comprising anucleic acid or polynucleotide of the invention. In some embodiments,the vector is an expression vector or expression cassette in which apolynucleotide sequence of the invention which encodes a variantprotease of the invention is operably linked to one or additionalnucleic acid segments required for efficient gene expression (e.g., apromoter operably linked to the polynucleotide of the invention whichencodes a variant protease of the invention). A vector may include atranscription terminator and/or a selection gene, such as an antibioticresistance gene that enables continuous cultural maintenance ofplasmid-infected host cells by growth in antimicrobial-containing media.

An expression vector may be derived from plasmid or viral DNA, or inalternative embodiments, contains elements of both. Exemplary vectorsinclude, but are not limited to pXX, pC 194, pJH 101, pE194, pHP13 (See,Harwood and Cutting [eds.], Chapter 3, Molecular Biological Methods forBacillus, John Wiley & Sons [1990]; suitable replicating plasmids for B.subtilis include those listed on p. 92; See also, Perego, IntegrationalVectors for Genetic Manipulations in Bacillus subtilis, in Sonenshein etal., [eds.] Bacillus subtilis and Other Gram-Positive Bacteria:Biochemistry, Physiology and Molecular Genetics, American Society forMicrobiology, Washington, D.C. [1993], pp. 615-624).

For expression and production of a protein of interest (e.g., variantprotease) in a cell, at least one expression vector comprising at leastone copy of a polynucleotide encoding the modified protease, andpreferably comprising multiple copies, is transformed into the cellunder conditions suitable for expression of the protease. In someembodiments of the present invention, a polynucleotide sequence encodingthe variant protease (as well as other sequences included in the vector)is integrated into the genome of the host cell, while in otherembodiments, a plasmid vector comprising a polynucleotide sequenceencoding the variant protease remains as autonomous extra-chromosomalelement within the cell. The invention provides both extrachromosomalnucleic acid elements as well as incoming nucleotide sequences that areintegrated into the host cell genome. The vectors described herein areuseful for production of the variant proteases of the invention. In someembodiments, a polynucleotide construct encoding the variant protease ispresent on an integrating vector that enables the integration andoptionally the amplification of the polynucleotide encoding the variantprotease into the bacterial chromosome. Examples of sites forintegration are well known to those skilled in the art. In someembodiments, transcription of a polynucleotide encoding a variantprotease of the invention is effectuated by a promoter that is thewild-type promoter for the selected precursor protease. In some otherembodiments, the promoter is heterologous to the precursor protease, butis functional in the host cell. Specifically, examples of suitablepromoters for use in bacterial host cells include, but are not limitedto, for example, the amyE, amyQ, amyL, pstS, sacB, pSPAC, pAprE, pVeg,pHpaII promoters, the promoter of the B. stearothermophilus maltogenicamylase gene, the B. amyloliquefaciens (BAN) amylase gene, the B.subtilis alkaline protease gene, the B. clausii alkaline protease genethe B. pumilis xylosidase gene, the B. thuringiensis cryIIIA, and the B.licheniformis alpha-amylase gene. Additional promoters include, but arenot limited to the A4 promoter, as well as phage Lambda P_(R) or P_(L)promoters, and the E. coli lac, trp or tac promoters.

Variant proteases of the present invention can be produced in host cellsof any suitable Gram-positive microorganism, including bacteria andfungi. For example, in some embodiments, the variant protease isproduced in host cells of fungal and/or bacterial origin. In someembodiments, the host cells are Bacillus sp., Streptomyces sp.,Escherichia sp. or Aspergillus sp. In some embodiments, the variantproteases are produced by Bacillus sp. host cells. Examples of Bacillussp. host cells that find use in the production of the variant proteasesof the invention include, but are not limited to B. licheniformis, B.lentus, B. subtilis, B. amyloliquefaciens, B. lentus, B. brevis, B.stearothermophilus, B. alkalophilus, B. coagulans, B. circulans, B.pumilis, B. thuringiensis, B. clausii, and B. megaterium, as well asother organisms within the genus Bacillus. In some embodiments, B.subtilis host cells are used for production of variant proteases. U.S.Pat. Nos. 5,264,366 and 4,760,025 (RE 34,606) describe various Bacillushost strains that can be used for producing variant proteases of theinvention, although other suitable strains can be used.

Several industrial bacterial strains that can be used to produce variantproteases of the invention include non-recombinant (i.e., wild-type)Bacillus sp. strains, as well as variants of naturally-occurring strainsand/or recombinant strains. In some embodiments, the host strain is arecombinant strain, wherein a polynucleotide encoding a polypeptide ofinterest has been introduced into the host. In some embodiments, thehost strain is a B. subtilis host strain and particularly a recombinantBacillus subtilis host strain. Numerous B. subtilis strains are known,including, but not limited to for example, 1A6 (ATCC 39085), 168 (1A01),SB19, W23, Ts85, B637, PB1753 through PB1758, PB3360, JH642, 1A243 (ATCC39,087), ATCC 21332, ATCC 6051, MI113, DE100 (ATCC 39,094), GX4931, PBT110, and PEP 211strain (See e.g., Hoch et al., Genetics 73:215-228[1973]; See also, U.S. Pat. Nos. 4,450,235 and 4,302,544, and EP0134048, each of which is incorporated by reference in its entirety).The use of B. subtilis as an expression host cells is well known in theart (See e.g., Palva et al., Gene 19:81-87 [1982]; Fahnestock andFischer, J. Bacteriol., 165:796-804 [1986]; and Wang et al., Gene69:39-47 [1988]).

In some embodiments, the Bacillus host cell is a Bacillus sp. thatincludes a mutation or deletion in at least one of the following genes,degU, degS, degR and degQ. Preferably the mutation is in a degU gene,and more preferably the mutation is degU(Hy)32 (See e.g., Msadek et al.,J. Bacteriol. 172:824-834 [1990]; and Olmos et al., Mol. Gen. Genet.253:562-567 [1997]). One suitable host strain is a Bacillus subtiliscarrying a degU32(Hy) mutation. In some embodiments, the Bacillus hostcomprises a mutation or deletion in scoC4 (See e.g., Caldwell et al., J.Bacteriol. 183:7329-7340 [2001]); spoIIE (See e.g., Arigoni et al., Mol.Microbiol. 31:1407-1415 [1999]); and/or oppA or other genes of the oppoperon (See e.g., Perego et al., Mol. Microbiol. 5:173-185 [1991]).Indeed, it is contemplated that any mutation in the opp operon thatcauses the same phenotype as a mutation in the oppA gene will find usein some embodiments of the altered Bacillus strain of the invention. Insome embodiments, these mutations occur alone, while in otherembodiments, combinations of mutations are present. In some embodiments,an altered Bacillus host cell strain that can be used to produce avariant protease of the invention is a Bacillus host strain that alreadyincludes a mutation in one or more of the above-mentioned genes. Inaddition, Bacillus sp. host cells that comprise mutation(s) and/ordeletions of endogenous protease genes find use. In some embodiments,the Bacillus host cell comprises a deletion of the aprE and the nprEgenes. In other embodiments, the Bacillus sp. host cell comprises adeletion of 5 protease genes, while in other embodiments, the Bacillussp. host cell comprises a deletion of 9 protease genes (See e.g., U.S.Pat. Appln. Pub. No. 2005/0202535, incorporated herein by reference).

Host cells are transformed with at least one nucleic acid encoding atleast one variant protease of the invention using any suitable methodknown in the art. Whether the nucleic acid is incorporated into a vectoror is used without the presence of plasmid DNA, it is typicallyintroduced into a microorganism, in some embodiments, preferably an E.coli cell or a competent Bacillus cell. Methods for introducing anucleic acid (e.g., DNA) into Bacillus cells or E. coli cells utilizingplasmid DNA constructs or vectors and transforming such plasmid DNAconstructs or vectors into such cells are well known. In someembodiments, the plasmids are subsequently isolated from E. coli cellsand transformed into Bacillus cells. However, it is not essential to useintervening microorganisms such as E. coli, and in some embodiments, aDNA construct or vector is directly introduced into a Bacillus host.

Those of skill in the art are well aware of suitable methods forintroducing nucleic acid or polynucleotide sequences of the inventioninto Bacillus cells (See e.g., Ferrari et al., “Genetics,” in Harwood etal. [eds.], Bacillus, Plenum Publishing Corp. [1989], pp. 57-72;Saunders et al., J. Bacteriol. 157:718-726 [1984]; Hoch et al., J.Bacteriol. 93:1925-1937 [1967]; Mann et al., Current Microbiol.13:131-135 [1986]; Holubova, Folia Microbiol. 30:97 [1985]; Chang etal., Mol. Gen. Genet. 168:11-115 [1979]; Vorobjeva et al., FEMSMicrobiol. Lett. 7:261-263 [1980]; Smith et al., Appl. Env. Microbiol.51:634 [1986]; Fisher et al., Arch. Microbiol. 139:213-217 [1981]; andMcDonald, J. Gen. Microbiol. 130:203 [1984]). Indeed, such methods astransformation, including protoplast transformation and congression,transduction, and protoplast fusion are well known and suited for use inthe present invention. Methods of transformation are used to introduce aDNA construct or vector comprising a nucleic acid encoding a variantprotease of the present invention into a host cell. Methods known in theart to transform Bacillus cells include such methods as plasmid markerrescue transformation, which involves the uptake of a donor plasmid bycompetent cells carrying a partially homologous resident plasmid (See,Contente et al., Plasmid 2:555-571 [1979]; Haima et al., Mol. Gen.Genet. 223:185-191 [1990]; Weinrauch et al., J. Bacteriol. 154:1077-1087[1983]; and Weinrauch et al., J. Bacteriol. 169:1205-1211 [1987]). Inthis method, the incoming donor plasmid recombines with the homologousregion of the resident “helper” plasmid in a process that mimicschromosomal transformation.

In addition to commonly used methods, in some embodiments, host cellsare directly transformed with a DNA construct or vector comprising anucleic acid encoding a variant protease of the invention (i.e., anintermediate cell is not used to amplify, or otherwise process, the DNAconstruct or vector prior to introduction into the host cell).Introduction of the DNA construct or vector of the invention into thehost cell includes those physical and chemical methods known in the artto introduce a nucleic acid sequence (e.g., DNA sequence) into a hostcell without insertion into a plasmid or vector. Such methods include,but are not limited to calcium chloride precipitation, electroporation,naked DNA, liposomes and the like. In additional embodiments, DNAconstructs or vector are co-transformed with a plasmid, without beinginserted into the plasmid. In further embodiments, a selective marker isdeleted from the altered Bacillus strain by methods known in the art(See, Stahl et al., J. Bacteriol. 158:411-418 [1984]; and Palmeros etal., Gene 247:255-264 [2000]).

In some embodiments, the transformed cells of the present invention arecultured in conventional nutrient media. The suitable specific cultureconditions, such as temperature, pH and the like are known to thoseskilled in the art and are well described in the scientific literature.In some embodiments, the invention provides a culture (e.g., cellculture) comprising at least one variant protease or at least onenucleic acid of the invention. Also provided are compositions comprisingat least one nucleic acid, vector, or DNA construct of the invention.

In some embodiments, host cells transformed with at least onepolynucleotide sequence encoding at least one variant protease of theinvention are cultured in a suitable nutrient medium under conditionspermitting the expression of the present protease, after which theresulting protease is recovered from the culture. The medium used toculture the cells comprises any conventional medium suitable for growingthe host cells, such as minimal or complex media containing appropriatesupplements. Suitable media are available from commercial suppliers ormay be prepared according to published recipes (See e.g., the cataloguesof the American Type Culture Collection). In some embodiments, theprotease produced by the cells is recovered from the culture medium byconventional procedures, including, but not limited to for example,separating the host cells from the medium by centrifugation orfiltration, precipitating the proteinaceous components of thesupernatant or filtrate by means of a salt (e.g., ammonium sulfate),chromatographic purification (e.g., ion exchange, gel filtration,affinity, etc.). Any method suitable for recovering or purifying avariant protease finds use in the present invention.

In some embodiments, a variant protease produced by a recombinant hostcell is secreted into the culture medium. A nucleic acid sequence thatencodes a purification facilitating domain may be used to facilitatepurification of soluble proteins. A vector or DNA construct comprising apolynucleotide sequence encoding a variant protease may further comprisea nucleic acid sequence encoding a purification facilitating domain tofacilitate purification of the variant protease (See e.g., Kroll et al.,DNA Cell Biol. 12:441-53 [1993]). Such purification facilitating domainsinclude, but are not limited to, for example, metal chelating peptidessuch as histidine-tryptophan modules that allow purification onimmobilized metals (See, Porath, Protein Expr. Purif. 3:263-281 [1992]),protein A domains that allow purification on immobilized immunoglobulin,and the domain utilized in the FLAGS extension/affinity purificationsystem (e.g., protein A domains available from Immunex Corp., Seattle,Wash.). The inclusion of a cleavable linker sequence such as Factor XAor enterokinase (e.g., sequences available from Invitrogen, San Diego,Calif.) between the purification domain and the heterologous proteinalso find use to facilitate purification.

Assays for detecting and measuring the enzymatic activity of an enzyme,such as a variant protease of the invention, are well known. Variousassays for detecting and measuring activity of proteases (e.g., variantproteases of the invention), are also known to those of ordinary skillin the art. In particular, assays are available for measuring proteaseactivity that are based on the release of acid-soluble peptides fromcasein or hemoglobin, measured as absorbance at 280 nm orcolorimetrically using the Folin method, well known to those skilled inthe art. Other exemplary assays involve the solubilization ofchromogenic substrates (See e.g., Ward, “Proteinases,” in Fogarty(ed.)., Microbial Enzymes and Biotechnology, Applied Science, London,[1983], pp. 251-317). Other exemplary assays include, but are notlimited to succinyl-Ala-Ala-Pro-Phe-para nitroanilide assay(suc-AAPF-pNA) and the 2,4,6-trinitrobenzene sulfonate sodium salt assay(TNBS assay). Numerous additional references known to those in the artprovide suitable methods (See e.g., Wells et al., Nucleic Acids Res.11:7911-7925 [1983]; Christianson et al., Anal. Biochem. 223:119-129[1994]; and Hsia et al., Anal Biochem. 242:221-227 [1999]).

A variety of methods can be used to determine the level of production ofa mature protease (e.g., mature variant proteases of the presentinvention) in a host cell. Such methods include, but are not limited to,for example, methods that utilize either polyclonal or monoclonalantibodies specific for the protease. Exemplary methods include, but arenot limited to enzyme-linked immunosorbent assays (ELISA),radioimmunoassays (RIA), fluorescent immunoassays (FIA), and fluorescentactivated cell sorting (FACS). These and other assays are well known inthe art (See e.g., Maddox et al., J. Exp. Med. 158:1211 [1983]).

In some other embodiments, the invention provides methods for making orproducing a mature variant protease of the invention. A mature variantprotease does not include a signal peptide or a propeptide sequence.Some methods comprise making or producing a variant protease of theinvention in a recombinant bacterial host cell, such as for example, aBacillus sp. cell (e.g., a B. subtilis cell). In some embodiments, theinvention provides a method of producing a variant protease of theinvention, the method comprising cultivating a recombinant host cellcomprising a recombinant expression vector comprising a nucleic acidencoding a variant protease of the invention under conditions conduciveto the production of the variant protease. Some such methods furthercomprise recovering the variant protease from the culture.

In some embodiments the invention provides methods of producing avariant protease of the invention, the methods comprising: (a)introducing a recombinant expression vector comprising a nucleic acidencoding a variant protease of the invention into a population of cells(e.g., bacterial cells, such as B. subtilis cells); and (b) culturingthe cells in a culture medium under conditions conducive to produce thevariant protease encoded by the expression vector. Some such methodsfurther comprise: (c) isolating the variant protease from the cells orfrom the culture medium.

Fabric and Home Care Products

In some embodiments, the protease variants, more specifically subtilisinvariants of the present invention can be used in compositions comprisingan adjunct material and a protease variant, wherein the composition is afabric and home care product.

In some embodiments, the fabric and home care product compositionscomprise at least one subtilisin variant, wherein said subtilisinvariant is a mature form having proteolytic activity and comprises anamino acid sequence comprising a combination of amino acid substitutionsat positions selected from List 1-19, and wherein amino acid positionsof the subtilisin variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO: 1.

The present invention further provides any of the above isolatedsubtilisin variants, wherein the subtilisin variant is a proteasevariant of Bacillus lentus subtilisin GG36 protease, wherein saidBacillus lentus subtilisin GG36 protease comprises the amino acidsequence shown in SEQ ID NO:2.

In some embodiments, the fabric and home care product compositionscomprise at least one subtilisin variant of Bacillus lentus subtilisinGG36 protease, wherein said Bacillus lentus subtilisin GG36 proteasecomprises the amino acid sequence shown in SEQ ID NO:2, and wherein saidGG36 protease is a mature form having proteolytic activity and comprisesan amino acid sequence comprising a combination of amino acidsubstitutions selected from List 1-19, and wherein amino acid positionsof the protease variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

In some embodiments, the fabric and home care product compositionscomprise any of the isolated subtilisin variants listed above, whereinthe total net charge of the variant is 0, +1, +2, +3, +4, +5, −1, −2,−3, −4, or −5 relative to the total net charge of the Bacillus lentussubtilisin GG36 protease. In some embodiments, the total net charge isobtained by one or more substitutions selected from: A001E, V004E,R010H/A, Q012E, A015D, N018D, R019H/S, V026D, K027E, N043D, R045C/T/S/P,S049D, V051E, G061E, N062D/E, N076D, N077D, S078D, S087D, A098E, S101D,S106E, G115E, G118D, N123D, S128D, P129E, S130D/E, S132D, A158E,G159D/E, S160D, S166D, R170T, N183D, N184D, N185E, R186H, S188D/E,A194E, A200D, Y209E, A215D, N204D, S212D, L217D/E, N218D, A230E, K235F,N237D, K237E, N238D, S240D, N243D, Q245D, R247L, N248D/E, K251C, N263D,N269D/E, A272D, A273E, R275H/S, A001R, V004R, Q012R, P014R, H017R,N018R/K, G020K/R, T022R/K, S024R/K, G025R, D032I, D041K/L/N, N043R/K,G046R, A048R, F050R/K, P055R, S056R/K, T057R, Q059K, G061R, N076K,S078R, P086R, S087R, E089G/P/I, G097R, S099R, Q109R, G115R, G118R,S132K, S144R, G159R/K, D181C/S/T, L196K, N204K, Q206R, K235R, Q236R/K,K237R, N238R, S240R, W241R, S242R/K, V244R, Q245R/K, N248R, H249R,N252R/K, S256R, G258R, T260K, N269R/K, or E271F/H/T/L/W/R/S/I/A/G/V, andwherein amino acid positions of the protease variant are numberedaccording to the numbering of corresponding amino acid positions in theamino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ shownin SEQ ID NO:1.

In some embodiments, the subtilisin variant(s) of the fabric and homecare product compositions are derived from a parent subtilisin that iscommercially available (e.g., SAVINASE®, POLARZYME®, KANNASE®,LIQUINASE®, LIQUINASE ULTRA®, SAVINASE ULTRA®, or OVOZYME® by NovozymesA/S); MAXACAL®, PROPERASE®, PURAFECT®, FN3®, FN4® and PURAFECT OXP®,PURAFAST™, PURAFECT® PRIME, or PURAMAX® by Genencor International) andthose available from Henkel/Kemira, namely BLAP (sequence shown in FIG.29 of U.S. Pat. No. 5,352,604 with the following mutationsS99D+S101R+S103A+V104I+G159S, hereinafter referred to as BLAP) and BLAPX (BLAP with S3T+V4I+V205I).

In some embodiments, the fabric and home care product compositionscomprise at least one protease variant whose parent has proteolyticactivity, wherein the variant protease comprises an amino acid sequencewhich differs from the amino acid sequence shown in SEQ ID NO:2 by nomore than 50, no more than 40, no more than 35, no more than 30, no morethan 25, no more than 20, no more than 19, no more than 18, no more than17, no more than 16, no more than 15, no more than 14, no more than 13,no more than 12, no more than 11, no more than 10, no more than 9, or nomore than 8 amino acid residues, wherein amino acid positions arenumbered according to the numbering of corresponding amino acidpositions in the amino acid sequence of Bacillus amyloliquefacienssubtilisin BPN′ shown in SEQ ID NO:1, as determined by alignment of thevariant protease amino acid sequence with the Bacillus amyloliquefacienssubtilisin BPN′ amino acid sequence.

Cleaning and/or Treatment Compositions

In some embodiments, the consumer product which comprises a cleaningand/or treatment composition, such as fabric and home care product,comprises at least one protease variant, particularly a subtilisinvariant and at least one adjunct material. In some embodiments, theseadjuncts are incorporated for example, to assist or enhance cleaningperformance, for treatment of the substrate to be cleaned, or to modifythe aesthetics of the cleaning composition as is the case with perfumes,colorants, dyes or the like. It is understood that such adjuncts are inaddition to the variant proteases of the present invention. The precisenature of these additional components, and levels of incorporationthereof, will depend on the physical form of the composition and thenature of the cleaning operation for which it is to be used. Thecleaning and/or treatment adjunct materials may be selected from one ormore from the list including, but not limited to, surfactants, builders,bleaches, bleach activators, bleach catalysts, other enzymes, enzymestabilizing systems, chelants, soil release polymers, dye transferagents, dispersants, suds suppressors, dyes, perfumes, colorants, fillersalts, hydrotropes, photoactivators, fluorescers, fabric conditioners,fabric softeners, hydrolyzable surfactants, preservatives, antioxidants,anti-shrinkage agents, anti-wrinkle agents, germicides, fungicides,color speckles, silvercare, anti-tarnish and/or anti-corrosion agents,alkalinity sources, solubilizing agents, carriers, processing aids,pigments, and pH control agents an encapsulate comprising a perfume, ahueing agent, surfactants, builders, chelating agents, dye transferinhibiting agents, dispersants, additional enzymes, enzyme stabilizers,catalytic materials, bleach activators, hydrogen peroxide, sources ofhydrogen peroxide, preformed peracids, polymeric dispersing agents, claysoil removal/anti-redeposition agents, brighteners, suds suppressors,dyes, perfumes, structure elasticizing agents, fabric softeners,hydrotropes, solvents and mixtures thereof (See e.g., U.S. Pat. Nos.6,610,642, 6,605,458, 5,705,464, 5,710,115, 5,698,504, 5,695,679,5,686,014 and 5,646,101, all of which are incorporated herein byreference). Embodiments of specific cleaning composition materials areexemplified in detail below. In embodiments in which the cleaning and/ortreatment adjunct materials are not compatible with the variantproteases of the present invention in the cleaning compositions, thensuitable methods of keeping the cleaning adjunct materials and theprotease(s) separated (i.e., not in contact with each other) untilcombination of the two components is appropriate are used. Suchseparation methods include any suitable method known in the art (e.g.,gelcaps, encapsulation, tablets, physical separation, etc.).

In some embodiments, cleaning and/or treatment composition, such asfabric and home care product, comprises at least one protease variantfurther comprise at least one additional non-immunoequivalent proteaseselected from subtilisins (EC 3.4.21.62); trypsin-like orchymotrypsin-like proteases; metalloproteases; and mixtures thereof.

In some embodiments, cleaning and/or treatment composition, such asfabric and home care product, comprises at least one protease variantfurther comprise at least one additional non-immunoequivalent proteaseselected from: subtilisins (EC 3.4.21.62) derived from B. subtilis, B.amyloliquefaciens, B. pumilus and B. gibsonii; trypsin proteases and/orchymotrypsin proteases derived from Cellulomonas; metalloproteasesderived from Bacillus amyloliquefaciens; and mixtures thereof.

In some embodiments, cleaning and/or treatment composition, such asfabric and home care product, comprises at least one protease variantfurther comprise at least one additional enzyme selected fromhemicellulases, peroxidases, proteases, cellulases, cellobiosedehydrogenases, xyloglucanases, xylanases, lipases, phospholipases,esterases, cutinases, pectinases, mannanases, pectate lyases,keratinases, reductases, oxidases, phenoloxidases, lipoxygenases,ligninases, pullulanases, tannases, pentosanases, lichenases glucanases,arabinosidases, hyaluronidases, chondroitinases, laccases, amylases, andmixtures thereof.

In some embodiments, cleaning and/or treatment composition, such asfabric and home care product, comprises at least one protease variantfurther comprise at least one additional enzyme selected from first-washlipases; alpha-amylases; bacterial cleaning cellulases; and mixturesthereof.

In some embodiments, cleaning and/or treatment composition, such asfabric and home care product, comprises at least one protease variantfurther comprise at least one of the following: an encapsulatecomprising a perfume comprises a perfume micro capsule; a hueing agentcomprising a material selected from basic, acid, hydrophobic, direct andpolymeric dyes, and dye-conjugates having a peak absorption wavelengthof from 550 nm to 650 nm and mixtures thereof; a detersive surfactantcomprising a material selected from anionic detersive surfactants,non-ionic detersive surfactant, cationic detersive surfactants,zwitterionic detersive surfactants and amphoteric detersive surfactantsand mixtures thereof; a builder comprising a material selected fromzeolites, phosphates and mixtures thereof; a silicate salt comprising amaterial selected from sodium silicate, potassium silicate and mixturesthereof; a brightener comprising a material selected from cold-watersoluble brighteners and mixtures thereof; a carboxylate polymercomprising a material selected from maleate/acrylate random copolymer orpolyacrylate homopolymer and mixtures thereof; a soil release polymercomprising a material selected from terephthalate co-polymers andmixtures thereof; a cellulosic polymer comprising a material selectedfrom alkyl cellulose, alkyl alkoxyalkyl cellulose, carboxyalkylcellulose, alkyl carboxyalkyl cellulose and mixtures thereof; a bleachcatalyst comprising a material selected from iminium cations, iminiumpolyions; iminium zwitterions; modified amines; modified amine oxides;N-sulphonyl imines; N-phosphonyl imines; N-acyl imines; thiadiazoledioxides; perfluoroimines; cyclic sugar ketones and mixtures thereof; ableach activator comprising a material selected from dodecanoyloxybenzene sulphonate, decanoyl oxybenzene sulphonate, decanoyloxybenzoic acid or salts thereof, 3,5,5-trimethyl hexanoyloxybenzenesulphonate, tetraacetyl ethylene diamine (TAED), nonanoyloxybenzenesulphonate (NOBS) and mixtures thereof; a source of hydrogen peroxidecomprising a material selected from inorganic perhydrate salts,including alkali metal salts such as sodium salts of perborate (usuallymono- or tetra-hydrate), percarbonate, persulphate, perphosphate,persilicate salts and mixtures thereof; a chelant comprising a materialselected from DTPA (diethylene triamine pentaacetic acid), HEDP(hydroxyethane diphosphonic acid), DTPMP (diethylene triaminepenta(methylene phosphonic acid)), ethylenediaminedisuccinic acid(EDDS), 1,2-dihydroxybenzene-3,5-disulfonic acid disodium salt hydrate,derivatives of said chelants; and mixtures thereof.

In some embodiments, cleaning and/or treatment composition, such asfabric and home care product, comprises at least one protease variantcomprise a fabric hueing agent selected from the group consisting ofdyes; dye-clay conjugates comprising at least one cationic-basic dye anda smectite clay; and mixtures thereof.

In some embodiments, cleaning and/or treatment composition, such asfabric and home care product, comprises at least one protease variantcomprise at least one fabric hueing agent selected from small moleculedyes and polymeric dyes and mixtures thereof optionally with a smectiteclay.

In some embodiments, cleaning and/or treatment composition, such asfabric and home care product, comprises at least one protease variantare provided in single or multiple-compartment unit doses. In someembodiments, the composition is a multi-compartment unit dose, whereinthe protease variant is in a different compartment than any source ofhydrogen peroxide and/or chelant and/or additional enzyme.

In some embodiments, cleaning and/or treatment composition, such asfabric and home care product, comprises at least one protease variantcomprise one or more of the following ingredients (based on totalcomposition weight): from about 0.0005 wt % to about 0.1 wt %, fromabout 0.001 wt % to about 0.05 wt %, or even from about 0.002 wt % toabout 0.03 wt % of said protease variant; and one or more of thefollowing: from about 0.00003 wt % to about 0.1 wt % fabric hueingagent; from about 0.001 wt % to about 5 wt %, perfume capsules; fromabout 0.001 wt % to about 1 wt %, cold-water soluble brighteners; fromabout 0.00003 wt % to about 0.1 wt % bleach catalysts; from about0.00003 wt % to about 0.1 wt % first wash lipases; from about 0.00003 wt% to about 0.1 wt % bacterial cleaning cellulases; and/or from about0.05 wt % to about 20 wt % Guerbet nonionic surfactants.

In some embodiments, cleaning and/or treatment composition, such asfabric and home care product, is a liquid laundry detergent, a dishwashing detergent.

It is intended that cleaning and/or treatment composition, such asfabric and home care product, is provided in any suitable form,including a fluid or solid. A cleaning and/or treatment composition,such as fabric and home care product, may be in the form of a unit dosepouch, especially when in the form of a liquid, and typically a cleaningand/or treatment composition, such as fabric and home care product, isat least partially, or even completely, enclosed by a water-solublepouch. In addition, in some embodiments of a cleaning and/or treatmentcomposition, such as fabric and home care product, comprising at leastone protease variant, a cleaning and/or treatment composition, such asfabric and home care product, may have any combination of parametersand/or characteristics detailed above. Unless otherwise noted, allcomponent or composition levels provided herein are made in reference tothe active level of that component or composition, and are exclusive ofimpurities, for example, residual solvents or by-products, which may bepresent in commercially available sources. Enzyme components weights arebased on total active protein. All percentages and ratios are calculatedby weight unless otherwise indicated. All percentages and ratios arecalculated based on the total composition unless otherwise indicated. Inthe exemplified detergent compositions, the enzymes levels are expressedby pure enzyme by weight of the total composition and unless otherwisespecified, the detergent ingredients are expressed by weight of thetotal compositions.

The cleaning compositions of the present invention are advantageouslyemployed for example, in laundry applications, hard surface cleaning,dishwashing applications, as well as cosmetic applications such asdentures, teeth, hair and skin. In addition, due to the uniqueadvantages of increased effectiveness in lower temperature solutions,the enzymes of the present invention are ideally suited for laundryapplications. Furthermore, the enzymes of the present invention find usein granular and liquid compositions.

The variant proteases of the present invention also find use in cleaningadditive products. In some embodiments, low temperature solutioncleaning applications find use. In some embodiments, the presentinvention provides cleaning additive products including at least oneenzyme of the present invention is ideally suited for inclusion in awash process when additional bleaching effectiveness is desired. Suchinstances include, but are not limited to low temperature solutioncleaning applications. In some embodiments, the additive product is inits simplest form, one or more proteases. In some embodiments, theadditive is packaged in dosage form for addition to a cleaning process.In some embodiments, the additive is packaged in dosage form foraddition to a cleaning process where a source of peroxygen is employedand increased bleaching effectiveness is desired. Any suitable singledosage unit form finds use with the present invention, including but notlimited to pills, tablets, gelcaps, or other single dosage units such aspre-measured powders or liquids. In some embodiments, filler(s) orcarrier material(s) are included to increase the volume of suchcompositions. Suitable filler or carrier materials include, but are notlimited to, various salts of sulfate, carbonate and silicate as well astalc, clay and the like. Suitable filler or carrier materials for liquidcompositions include, but are not limited to water or low molecularweight primary and secondary alcohols including polyols and diols.Examples of such alcohols include, but are not limited to, methanol,ethanol, propanol and isopropanol. In some embodiments, the compositionscontain from about 5% to about 90% of such materials. Acidic fillersfind use to reduce pH. Alternatively, in some embodiments, the cleaningadditive includes adjunct ingredients, as more fully described below.

The present cleaning compositions and cleaning additives require aneffective amount of at least one of the protease variants, morespecifically subtilisin variants provided herein, alone or incombination with other proteases and/or additional enzymes. The requiredlevel of enzyme is achieved by the addition of one or more proteasevariants, more specifically subtilisin variants of the presentinvention. Typically the present cleaning compositions comprise at leastabout 0.0001 weight percent, from about 0.0001 to about 10, from about0.001 to about 1, or even from about 0.01 to about 0.1 weight percent ofat least one of the variant proteases of the present invention.

The cleaning compositions herein are typically formulated such that,during use in aqueous cleaning operations, the wash water will have a pHof from about 5.0 to about 11.5 or even from about 7.5 to about 10.5.Liquid product formulations are typically formulated to have a neat pHfrom about 3.0 to about 9.0 or even from about 3 to about 5. Granularlaundry products are typically formulated to have a pH from about 9 toabout 11. Techniques for controlling pH at recommended usage levelsinclude the use of buffers, alkalis, acids, etc., and are well known tothose skilled in the art.

Suitable “low pH cleaning compositions” typically have a neat pH of fromabout 3 to about 5, and are typically free of surfactants that hydrolyzein such a pH environment. Such surfactants include sodium alkyl sulfatesurfactants that comprise at least one ethylene oxide moiety or evenfrom about 1 to about 16 moles of ethylene oxide. Such cleaningcompositions typically comprise a sufficient amount of a pH modifier,such as sodium hydroxide, monoethanolamine or hydrochloric acid, toprovide such cleaning composition with a neat pH of from about 3 toabout 5. Such compositions typically comprise at least one acid stableenzyme. In some embodiments, the compositions are liquids, while inother embodiments, they are solids. The pH of such liquid compositionsis typically measured as a neat pH. The pH of such solid compositions ismeasured as a 10% solids solution of said composition wherein thesolvent is distilled water. In these embodiments, all pH measurementsare taken at 20° C., unless otherwise indicated.

In some embodiments, when the variant protease(s) is/are employed in agranular composition or liquid, it is desirable for the variant proteaseto be in the form of an encapsulated particle to protect the variantprotease from other components of the granular composition duringstorage. In addition, encapsulation is also a means of controlling theavailability of the variant protease during the cleaning process. Insome embodiments, encapsulation enhances the performance of the variantprotease(s) and/or additional enzymes. In this regard, the variantproteases of the present invention are encapsulated with any suitableencapsulating material known in the art. In some embodiments, theencapsulating material typically encapsulates at least part of thecatalyst for the variant protease(s) of the present invention.Typically, the encapsulating material is water-soluble and/orwater-dispersible. In some embodiments, the encapsulating material has aglass transition temperature (Tg) of 0° C. or higher. Glass transitiontemperature is described in more detail in WO 97/11151. Theencapsulating material is typically selected from consisting ofcarbohydrates, natural or synthetic gums, chitin, chitosan, celluloseand cellulose derivatives, silicates, phosphates, borates, polyvinylalcohol, polyethylene glycol, paraffin waxes, and combinations thereof.When the encapsulating material is a carbohydrate, it is typicallyselected from monosaccharides, oligosaccharides, polysaccharides, andcombinations thereof. In some typical embodiments, the encapsulatingmaterial is a starch (See e.g., EP 0 922 499; U.S. Pat. No. 4,977,252;U.S. Pat. No. 5,354,559, and U.S. Pat. No. 5,935,826). In someembodiments, the encapsulating material is a microsphere made fromplastic such as thermoplastics, acrylonitrile, methacrylonitrile,polyacrylonitrile, polymethacrylonitrile and mixtures thereof;commercially available microspheres that find use include, but are notlimited to those supplied by EXPANCEL® (Stockviksverken, Sweden), and PM6545, PM 6550, PM 7220, PM 7228, EXTENDOSPHERES®, LUXSIL®, Q-CEL®, andSPHERICEL® (PQ Corp., Valley Forge, Pa.).

As described herein, the variant proteases of the present invention findparticular use in the cleaning industry, including, but not limited tolaundry and dish detergents. These applications place enzymes undervarious environmental stresses. The variant proteases of the presentinvention provide advantages over many currently used enzymes, due totheir stability under various conditions.

Indeed, there are a variety of wash conditions including varyingdetergent formulations, wash water volumes, wash water temperatures, andlengths of wash time, to which proteases involved in washing areexposed. In addition, detergent formulations used in differentgeographical areas have different concentrations of their relevantcomponents present in the wash water. For example, European detergentstypically have about 4500-5000 ppm of detergent components in the washwater, while Japanese detergents typically have approximately 667 ppm ofdetergent components in the wash water. In North America, particularlythe United States, detergents typically have about 975 ppm of detergentcomponents present in the wash water.

A low detergent concentration system includes detergents where less thanabout 800 ppm of the detergent components are present in the wash water.Japanese detergents are typically considered low detergent concentrationsystem as they have approximately 667 ppm of detergent componentspresent in the wash water.

A medium detergent concentration includes detergents where between about800 ppm and about 2000 ppm of the detergent components are present inthe wash water. North American detergents are generally considered to bemedium detergent concentration systems as they have approximately 975ppm of detergent components present in the wash water. Brazil typicallyhas approximately 1500 ppm of detergent components present in the washwater.

A high detergent concentration system includes detergents where greaterthan about 2000 ppm of the detergent components are present in the washwater. European detergents are generally considered to be high detergentconcentration systems as they have approximately 4500-5000 ppm ofdetergent components in the wash water.

Latin American detergents are generally high suds phosphate builderdetergents and the range of detergents used in Latin America can fall inboth the medium and high detergent concentrations as they range from1500 ppm to 6000 ppm of detergent components in the wash water. Asmentioned above, Brazil typically has approximately 1500 ppm ofdetergent components present in the wash water. However, other high sudsphosphate builder detergent geographies, not limited to other LatinAmerican countries, may have high detergent concentration systems up toabout 6000 ppm of detergent components present in the wash water.

In light of the foregoing, it is evident that concentrations ofdetergent compositions in typical wash solutions throughout the worldvaries from less than about 800 ppm of detergent composition (“lowdetergent concentration geographies”), for example about 667 ppm inJapan, to between about 800 ppm to about 2000 ppm (“medium detergentconcentration geographies”), for example about 975 ppm in U.S. and about1500 ppm in Brazil, to greater than about 2000 ppm (“high detergentconcentration geographies”), for example about 4500 ppm to about 5000ppm in Europe and about 6000 ppm in high suds phosphate buildergeographies.

The concentrations of the typical wash solutions are determinedempirically. For example, in the U.S., a typical washing machine holds avolume of about 64.4 L of wash solution. Accordingly, in order to obtaina concentration of about 975 ppm of detergent within the wash solutionabout 62.79 g of detergent composition must be added to the 64.4 L ofwash solution. This amount is the typical amount measured into the washwater by the consumer using the measuring cup provided with thedetergent.

As a further example, different geographies use different washtemperatures. The temperature of the wash water in Japan is typicallyless than that used in Europe. For example, the temperature of the washwater in North America and Japan is typically between about 10 and about30° C. (e.g., about 20° C.), whereas the temperature of wash water inEurope is typically between about 30 and about 60° C. (e.g., about 40°C.). However, in the interest of saving energy, many consumers areswitching to using cold water washing. In addition, in some furtherregions, cold water is typically used for laundry, as well as dishwashing applications. In some embodiments, the “cold water washing” ofthe present invention utilizes “cold water detergent” suitable forwashing at temperatures from about 10° C. to about 40° C., or from about20° C. to about 30° C., or from about 15° C. to about 25° C., as well asall other combinations within the range of about 15° C. to about 35° C.,and all ranges within 10° C. to 40° C.

As a further example, different geographies typically have differentwater hardness. Water hardness is usually described in terms of thegrains per gallon mixed Ca²⁺/Mg²⁺. Hardness is a measure of the amountof calcium (Ca²⁺) and magnesium (Mg²⁺) in the water. Most water in theUnited States is hard, but the degree of hardness varies. Moderatelyhard (60-120 ppm) to hard (121-181 ppm) water has 60 to 181 parts permillion (parts per million converted to grains per U.S. gallon is ppm #divided by 17.1 equals grains per gallon) of hardness minerals.

Water Grains per gallon Parts per million Soft less than 1.0 less than17 Slightly hard 1.0 to 3.5 17 to 60 Moderately hard 3.5 to 7.0  60 to120 Hard  7.0 to 10.5 120 to 180 Very hard greater than 10.5 greaterthan 180

European water hardness is typically greater than about 10.5 (forexample about 10.5 to about 20.0) grains per gallon mixed Ca²⁺/Mg²⁺(e.g., about 15 grains per gallon mixed Ca²⁺/Mg²⁺). North American waterhardness is typically greater than Japanese water hardness, but lessthan European water hardness. For example, North American water hardnesscan be between about 3 to about 10 grains, about 3 to about 8 grains orabout 6 grains. Japanese water hardness is typically lower than NorthAmerican water hardness, usually less than about 4, for example about 3grains per gallon mixed Ca²⁺/Mg²⁺.

Accordingly, in some embodiments, the present invention provides variantproteases that show surprising wash performance in at least one set ofwash conditions (e.g., water temperature, water hardness, and/ordetergent concentration). In some embodiments, the variant proteases ofthe present invention are comparable in wash performance to othersubtilisin proteases. In some embodiments, the variant proteases of thepresent invention exhibit enhanced wash performance as compared tosubtilisin proteases currently commercially available. Thus, in someembodiments of the present invention, the variant proteases providedherein exhibit enhanced oxidative stability, enhanced thermal stability,enhanced cleaning capabilities under various conditions, and/or enhancedchelator stability. In addition, the variant proteases of the presentinvention find use in cleaning compositions that do not includedetergents, again either alone or in combination with builders andstabilizers.

In some embodiments of the present invention, the cleaning compositionscomprise at least one variant protease of the present invention at alevel from about 0.00001% to about 10% by weight of the composition andthe balance (e.g., about 99.999% to about 90.0%) comprising cleaningadjunct materials by weight of composition. In some other embodiments ofthe present invention, the cleaning compositions of the presentinvention comprises at least one variant protease at a level of about0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about2%, about 0.005% to about 0.5% by weight of the composition and thebalance of the cleaning composition (e.g., about 99.9999% to about90.0%, about 99.999% to about 98%, about 99.995% to about 99.5% byweight) comprising cleaning adjunct materials.

In some embodiments, the cleaning compositions of the present inventioncomprise one or more additional detergent enzymes, which providecleaning performance and/or fabric care and/or dishwashing benefits.Examples of suitable enzymes include, but are not limited to,hemicellulases, cellulases, peroxidases, proteases, xylanases, lipases,phospholipases, esterases, cutinases, pectinases, pectate lyases,mannanases, keratinases, reductases, oxidases, phenoloxidases,lipoxygenases, ligninases, pullulanases, tannases, pentosanases,malanases, β-glucanases, arabinosidases, hyaluronidases,chondroitinases, laccases, and amylases, or any combinations or mixturesthereof. In some embodiments, a combination of enzymes is used (i.e., a“cocktail”) comprising conventional applicable enzymes like protease,lipase, cutinase and/or cellulase in conjunction with amylase is used.

In addition to the protease variants, more specifically subtilisinvariants provided herein, any other suitable protease finds use in thecompositions of the present invention. Suitable proteases include thoseof animal, vegetable or microbial origin. In some embodiments, microbialproteases are used. In some embodiments, chemically or geneticallymodified mutants are included. In some embodiments, the protease is aserine protease, preferably an alkaline microbial protease or atrypsin-like protease. Examples of alkaline proteases includesubtilisins, especially those derived from Bacillus (e.g., subtilisin,lentus, amyloliquefaciens, subtilisin Carlsberg, subtilisin 309,subtilisin 147 and subtilisin 168). Additional examples include thosemutant proteases described in U.S. Pat. Nos. RE 34,606, 5,955,340,5,700,676, 6,312,936, and 6,482,628, all of which are incorporatedherein by reference. Additional protease examples include, but are notlimited to trypsin (e.g., of porcine or bovine origin), and the Fusariumprotease described in WO 89/06270. In some embodiments, commerciallyavailable protease enzymes that find use in the present inventioninclude, but are not limited to MAXATASE®, MAXACAL™, MAXAPEM™,OPTICLEAN®, OPTIMASE®, PROPERASE®, PURAFECT®, PURAFECT® OXP, PURAMAX™,EXCELLASE™, and PURAFAST™ (GenenCor); ALCALASE®, SAVINASE®, PRIMASE®,DURAZYM™, POLARZYME®, OVOZYME®, KANNASE®, LIQUANASE®, NEUTRASE®, RELASE®and ESPERASE® (Novozymes); BLAP™ and BLAP™ variants (HenkelKommanditgesellschaft auf Aktien, Duesseldorf, Germany), and KAP (B.alkalophilus subtilisin; Kao Corp., Tokyo, Japan). Various proteases aredescribed in WO95/23221, WO 92/21760, U.S. Pat. Publ. No. 2008/0090747,and U.S. Pat. Nos. 5,801,039, 5,340,735, 5,500,364, 5,855,625, U.S. RE34,606, U.S. Pat. Nos. 5,955,340, 5,700,676, 6,312,936, and 6,482,628,and various other patents. In some further embodiments, metalloproteasesfind use in the present invention, including but not limited to theneutral metalloprotease described in WO 07/044,993.

In addition, any suitable lipase finds use in the present invention.Suitable lipases include, but are not limited to those of bacterial orfungal origin. Chemically or genetically modified mutants areencompassed by the present invention. Examples of useful lipases includeHumicola lanuginosa lipase (See e.g., EP 258 068, and EP 305 216),Rhizomucor miehei lipase (See e.g., EP 238 023), Candida lipase, such asC. antarctica lipase (e.g., the C. antarctica lipase A or B; See e.g.,EP 214 761), Pseudomonas lipases such as P. alcaligenes lipase and P.pseudoalcaligenes lipase (See e.g., EP 218 272), P. cepacia lipase (Seee.g., EP 331 376), P. stutzeri lipase (See e.g., GB 1,372,034), P.fluorescens lipase, Bacillus lipase (e.g., B. subtilis lipase [Dartoiset al., Biochem. Biophys. Acta 1131:253-260 [1993]); B.stearothermophilus lipase [See e.g., JP 64/744992]; and B. pumiluslipase [See e.g., WO 91/16422]).

Furthermore, a number of cloned lipases find use in some embodiments ofthe present invention, including but not limited to Penicilliumcamembertii lipase (See, Yamaguchi et al., Gene 103:61-67 [1991]),Geotricum candidum lipase (See, Schimada et al., J. Biochem.,106:383-388 [1989]), and various Rhizopus lipases such as R. delemarlipase (See, Hass et al., Gene 109:117-113 [1991]), a R. niveus lipase(Kugimiya et al., Biosci. Biotech. Biochem. 56:716-719 [1992]) and R.oryzae lipase.

Other types of lipolytic enzymes such as cutinases also find use in someembodiments of the present invention, including but not limited to thecutinase derived from Pseudomonas mendocina (See, WO 88/09367), and thecutinase derived from Fusarium solani pisi (See, WO 90/09446).

Additional suitable lipases include commercially available lipases suchas M1 LIPASE™, LUMA FAST™, and LIPOMAX™ (Genencor); LIPEX®, LIPOLASE®and LIPOLASE® ULTRA (Novozymes); and LIPASE P™ “Amano” (AmanoPharmaceutical Co. Ltd., Japan).

In some embodiments of the present invention, the cleaning compositionsof the present invention further comprise lipases at a level from about0.00001% to about 10% of additional lipase by weight of the compositionand the balance of cleaning adjunct materials by weight of composition.In some other embodiments of the present invention, the cleaningcompositions of the present invention also comprise lipases at a levelof about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% toabout 2%, about 0.005% to about 0.5% lipase by weight of thecomposition.

In some embodiments of the present invention, any suitable amylase findsuse in the present invention. In some embodiments, any amylase (e.g.,alpha and/or beta) suitable for use in alkaline solutions also find use.Suitable amylases include, but are not limited to those of bacterial orfungal origin. Chemically or genetically modified mutants are includedin some embodiments. Amylases that find use in the present invention,include, but are not limited to α-amylases obtained from B.licheniformis (See e.g., GB 1,296,839). Commercially available amylasesthat find use in the present invention include, but are not limited toDURAMYL®, TERMAMYL®, FUNGAMYL®, STAINZYME®, STAINZYME PLUS®, STAINZYMEULTRA®, and BAN™ (Novozymes), as well as POWERASE™, RAPIDASE® andMAXAMYL® P (Genencor).

In some embodiments of the present invention, the cleaning compositionsof the present invention further comprise amylases at a level from about0.00001% to about 10% of additional amylase by weight of the compositionand the balance of cleaning adjunct materials by weight of composition.In some other embodiments of the present invention, the cleaningcompositions of the present invention also comprise amylases at a levelof about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% toabout 2%, about 0.005% to about 0.5% amylase by weight of thecomposition.

In some further embodiments, any suitable cellulase finds used in thecleaning compositions of the present invention. Suitable cellulasesinclude, but are not limited to those of bacterial or fungal origin.Chemically or genetically modified mutants are included in someembodiments. Suitable cellulases include, but are not limited toHumicola insolens cellulases (See e.g., U.S. Pat. No. 4,435,307).Especially suitable cellulases are the cellulases having color carebenefits (See e.g., EP 0 495 257). Commercially available cellulasesthat find use in the present include, but are not limited to CELLUZYME®,CAREZYME® (Novozymes), and KAC-500(B)™ (Kao Corporation). In someembodiments, cellulases are incorporated as portions or fragments ofmature wild-type or variant cellulases, wherein a portion of theN-terminus is deleted (See e.g., U.S. Pat. No. 5,874,276). In someembodiments, the cleaning compositions of the present invention furthercomprise cellulases at a level from about 0.00001% to about 10% ofadditional cellulase by weight of the composition and the balance ofcleaning adjunct materials by weight of composition. In some otherembodiments of the present invention, the cleaning compositions of thepresent invention also comprise cellulases at a level of about 0.0001%to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, about0.005% to about 0.5% cellulase by weight of the composition.

Any mannanase suitable for use in detergent compositions also finds usein the present invention. Suitable mannanases include, but are notlimited to those of bacterial or fungal origin. Chemically orgenetically modified mutants are included in some embodiments. Variousmannanases are known which find use in the present invention (See e.g.,U.S. Pat. No. 6,566,114, U.S. Pat. No. 6,602,842, and U.S. Pat. No.6,440,991, all of which are incorporated herein by reference). In someembodiments, the cleaning compositions of the present invention furthercomprise mannanases at a level from about 0.00001% to about 10% ofadditional mannanase by weight of the composition and the balance ofcleaning adjunct materials by weight of composition. In some embodimentsof the present invention, the cleaning compositions of the presentinvention also comprise mannanases at a level of about 0.0001% to about10%, about 0.001% to about 5%, about 0.001% to about 2%, about 0.005% toabout 0.5% mannanase by weight of the composition.

In some embodiments, peroxidases are used in combination with hydrogenperoxide or a source thereof (e.g., a percarbonate, perborate orpersulfate) in the compositions of the present invention. In somealternative embodiments, oxidases are used in combination with oxygen.Both types of enzymes are used for “solution bleaching” (i.e., toprevent transfer of a textile dye from a dyed fabric to another fabricwhen the fabrics are washed together in a wash liquor), preferablytogether with an enhancing agent (See e.g., WO 94/12621 and WO95/01426). Suitable peroxidases/oxidases include, but are not limited tothose of plant, bacterial or fungal origin. Chemically or geneticallymodified mutants are included in some embodiments. In some embodiments,the cleaning compositions of the present invention further compriseperoxidase and/or oxidase enzymes at a level from about 0.00001% toabout 10% of additional peroxidase and/or oxidase by weight of thecomposition and the balance of cleaning adjunct materials by weight ofcomposition. In some other embodiments of the present invention, thecleaning compositions of the present invention also comprise, peroxidaseand/or oxidase enzymes at a level of about 0.0001% to about 10%, about0.001% to about 5%, about 0.001% to about 2%, about 0.005% to about 0.5%peroxidase and/or oxidase enzymes by weight of the composition.

In some embodiments, additional enzymes find use, including but notlimited to perhydrolases (See e.g., WO 05/056782). In addition, in someembodiments, mixtures of the above mentioned enzymes are encompassedherein, in particular one or more additional protease, amylase, lipase,mannanase, and/or at least one cellulase. Indeed, it is contemplatedthat various mixtures of these enzymes will find use in the presentinvention. It is also contemplated that the varying levels of thevariant protease(s) and one or more additional enzymes may bothindependently range to about 10%, the balance of the cleaningcomposition being cleaning adjunct materials. The specific selection ofcleaning adjunct materials are readily made by considering the surface,item, or fabric to be cleaned, and the desired form of the compositionfor the cleaning conditions during use (e.g., through the wash detergentuse).

In some embodiments, an effective amount of one or more variantprotease(s) provided herein is included in compositions useful forcleaning a variety of surfaces in need of proteinaceous stain removal.Such cleaning compositions include cleaning compositions for suchapplications as cleaning hard surfaces, fabrics, and dishes. Indeed, insome embodiments, the present invention provides fabric cleaningcompositions, while in other embodiments, the present invention providesnon-fabric cleaning compositions. Notably, the present invention alsoprovides cleaning compositions suitable for personal care, includingoral care (including dentrifices, toothpastes, mouthwashes, etc., aswell as denture cleaning compositions), skin, and hair cleaningcompositions. It is intended that the present invention encompassdetergent compositions in any form (i.e., liquid, granular, bar,semi-solid, gels, emulsions, tablets, capsules, etc.).

By way of example, several cleaning compositions wherein the variantproteases of the present invention find use are described in greaterdetail below. In some embodiments in which the cleaning compositions ofthe present invention are formulated as compositions suitable for use inlaundry machine washing method(s), the compositions of the presentinvention preferably contain at least one surfactant and at least onebuilder compound, as well as one or more cleaning adjunct materialspreferably selected from organic polymeric compounds, bleaching agents,additional enzymes, suds suppressors, dispersants, lime-soapdispersants, soil suspension and anti-redeposition agents and corrosioninhibitors. In some embodiments, laundry compositions also containsoftening agents (i.e., as additional cleaning adjunct materials). Thecompositions of the present invention also find use detergent additiveproducts in solid or liquid form. Such additive products are intended tosupplement and/or boost the performance of conventional detergentcompositions and can be added at any stage of the cleaning process. Insome embodiments, the density of the laundry detergent compositionsherein ranges from about 400 to about 1200 g/liter, while in otherembodiments, it ranges from about 500 to about 950 g/liter ofcomposition measured at 20° C.

In embodiments formulated as compositions for use in manual dishwashingmethods, the compositions of the invention preferably contain at leastone surfactant and preferably at least one additional cleaning adjunctmaterial selected from organic polymeric compounds, suds enhancingagents, group II metal ions, solvents, hydrotropes and additionalenzymes.

In some embodiments, various cleaning compositions such as thoseprovided in U.S. Pat. No. 6,605,458, find use with the variant proteasesof the present invention. Thus, in some embodiments, the compositionscomprising at least one variant protease of the present invention is acompact granular fabric cleaning composition, while in otherembodiments, the composition is a granular fabric cleaning compositionuseful in the laundering of colored fabrics, in further embodiments, thecomposition is a granular fabric cleaning composition which providessoftening through the wash capacity, in additional embodiments, thecomposition is a heavy duty liquid fabric cleaning composition. In someembodiments, the compositions comprising at least one variant proteaseof the present invention are fabric cleaning compositions such as thosedescribed in U.S. Pat. Nos. 6,610,642 and 6,376,450. In addition, thevariant proteases of the present invention find use in granular laundrydetergent compositions of particular utility under European or Japanesewashing conditions (See e.g., U.S. Pat. No. 6,610,642).

In some alternative embodiments, the present invention provides hardsurface cleaning compositions comprising at least one variant proteaseprovided herein. Thus, in some embodiments, the compositions comprisingat least one variant protease of the present invention is a hard surfacecleaning composition such as those described in U.S. Pat. Nos.6,610,642, 6,376,450, and 6,376,450.

In yet further embodiments, the present invention provides dishwashingcompositions comprising at least one variant protease provided herein.Thus, in some embodiments, the compositions comprising at least onevariant protease of the present invention is a hard surface cleaningcomposition such as those in U.S. Pat. Nos. 6,610,642 and 6,376,450. Insome still further embodiments, the present invention providesdishwashing compositions comprising at least one variant proteaseprovided herein. In some further embodiments, the compositionscomprising at least one variant protease of the present inventioncomprise oral care compositions such as those in U.S. Pat. Nos.6,376,450, and 6,376,450. The formulations and descriptions of thecompounds and cleaning adjunct materials contained in the aforementionedU.S. Pat. Nos. 6,376,450, 6,605,458, 6,605,458, and 6,610,642, find usewith the variant proteases provided herein.

The cleaning compositions of the present invention are formulated intoany suitable form and prepared by any process chosen by the formulator,non-limiting examples of which are described in U.S. Pat. Nos.5,879,584, 5,691,297, 5,574,005, 5,569,645, 5,565,422, 5,516,448,5,489,392, and 5,486,303, all of which are incorporated herein byreference. When a low pH cleaning composition is desired, the pH of suchcomposition is adjusted via the addition of a material such asmonoethanolamine or an acidic material such as HCl.

The cleaning compositions disclosed herein of find use in cleaning asitus (e.g., a surface, item, dishware, or fabric). Typically, at leasta portion of the situs is contacted with an embodiment of the presentcleaning composition, in neat form or diluted in a wash liquor, and thenthe situs is optionally washed and/or rinsed. For purposes of thepresent invention, “washing” includes but is not limited to, scrubbing,and mechanical agitation. In some embodiments, the cleaning compositionsare typically employed at concentrations of from about 500 ppm to about15,000 ppm in solution. When the wash solvent is water, the watertemperature typically ranges from about 5° C. to about 90° C. and, whenthe situs comprises a fabric, the water to fabric mass ratio istypically from about 1:1 to about 30:1.

More detail regarding suitable cleaning and/or treatment adjuncts isgiven below.

Fabric Hueing Agents.

Although it is not preferred to incorporate additional fabric shadingdyes, in addition to the thiophene azo dye, the composition may compriseone or more additional fabric hueing agents. Suitable fabric hueingagents include dyes, dye-clay conjugates, and pigments. Suitable dyesinclude those that deposit more onto cotton textiles compared todeposition onto synthetic textiles such as polyester and/or nylon.Further suitable dyes include those that deposit more onto syntheticfibres such as polyester and/or nylon compared to cotton. Suitable dyesinclude small molecule dyes and polymeric dyes. Suitable small moleculedyes include small molecule dyes selected from the group consisting ofdyes falling into the Colour Index (C.I.) classifications of DirectBlue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, BasicBlue, Basic Violet and Basic Red, or mixtures thereof. Examples of smallmolecule dyes include those selected from the group consisting of ColourIndex (Society of Dyers and Colourists, Bradford, UK) numbers DirectViolet 9, Direct Violet 35, Direct Violet 48, Direct Violet 51, DirectViolet 66, Direct Violet 99, Direct Blue 1, Direct Blue 71, Direct Blue80, Direct Blue 279, Acid Red 17, Acid Red 73, Acid Red 88, Acid Red150, Acid Violet 15, Acid Violet 17, Acid Violet 24, Acid Violet 43,Acid Red 52, Acid Violet 49, Acid Violet 50, Acid Blue 15, Acid Blue 17,Acid Blue 25, Acid Blue 29, Acid Blue 40, Acid Blue 45, Acid Blue 75,Acid Blue 80, Acid Blue 83, Acid Blue 90 and Acid Blue 113, Acid Black1, Basic Violet 1, Basic Violet 3, Basic Violet 4, Basic Violet 10,Basic Violet 35, Basic Blue 3, Basic Blue 16, Basic Blue 22, Basic Blue47, Basic Blue 66, Basic Blue 75, Basic Blue 159, small molecule dyesselected from the group consisting of Colour Index (Society of Dyers andColourists, Bradford, UK) numbers Acid Violet 17, Acid Violet 43, AcidRed 52, Acid Red 73, Acid Red 88, Acid Red 150, Acid Blue 25, Acid Blue29, Acid Blue 45, Acid Blue 113, Acid Black 1, Direct Blue 1, DirectBlue 71. Direct Violet small molecule dyes may be preferred. Dyesselected from the group consisting Acid Violet 17, Direct Blue 71,Direct Violet 51, Direct Blue 1, Acid Red 88, Acid Red 150, Acid Blue29, Acid Blue 113 and mixtures thereof may be preferred.

Suitable polymeric dyes include polymeric dyes selected from the groupconsisting of polymers containing covalently bound chromogens(dye-polymer conjugates) and polymers with chromogens co-polymerizedinto the backbone of the polymer and mixtures thereof, and polymericdyes selected from the group consisting of fabric-substantive colorantssold under the name of Liquitint® (Milliken, Spartanburg, S.C., USA),dye-polymer conjugates formed from at least one reactive dye and apolymer selected from the group consisting of polymers comprising amoiety selected from the group consisting of a hydroxyl moiety, aprimary amine moiety, a secondary amine moiety, a thiol moiety andmixtures thereof. In still another aspect, suitable polymeric dyesinclude polymeric dyes selected from the group consisting of Liquitint®(Milliken, Spartanburg, S.C., USA) Violet CT, carboxymethyl cellulose(CMC) conjugated with a reactive blue, reactive violet or reactive reddye such as CMC conjugated with C.I. Reactive Blue 19, sold by Megazyme,Wicklow, Ireland under the product name AZO-CM-CELLULOSE, product codeS-ACMC, alkoxylated triphenyl-methane polymeric colourants, alkoxylatedthiophene polymeric colourants, and mixtures thereof. Preferredadditional hueing dyes include the whitening agents found in WO 08/87497A1. These whitening agents may be characterized by the followingstructure (I):

wherein R₁ and R₂ can independently be selected from:

a) [(CH₂CR′HO)_(x)(CH₂CR″HO)_(y)H], wherein R′ is selected from thegroup consisting of H, CH₃, CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof;wherein R″ is selected from the group consisting of H,CH₂O(CH₂CH₂O)_(y)H, and mixtures thereof; wherein x+y≦5; wherein y≧1;and wherein z=0 to 5;

b) R₁=alkyl, aryl or aryl alkyl and R₂═[(CH₂CR′HO)(CH₂CR″HO)_(y)H]

wherein R′ is selected from the group consisting of H, CH₃,CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof; wherein R″ is selected fromthe group consisting of H, CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof;wherein x+y≦10; wherein y≧1; and wherein z=0 to 5;

c) R₁═[CH₂CH(OR₃)CH₂OR₄] and R₂═[CH₂CH(OR₃)CH₂OR₄]

wherein R₃ is selected from the group consisting of H, (CH₂CH₂O)_(z)H,and mixtures thereof; and wherein z=0 to 10;

wherein R₄ is selected from the group consisting of (C₁-C₁₆)alkyl, arylgroups, and mixtures thereof; and

d) wherein R1 and R2 can independently be selected from the aminoaddition product of styrene oxide, glycidyl methyl ether, isobutylglycidyl ether, isopropylglycidyl ether, t-butyl glycidyl ether,2-ethylhexylgycidyl ether, and glycidylhexadecyl ether, followed by theaddition of from 1 to 10 alkylene oxide units.

A preferred additional fabric hueing agent which may be incorporatedinto the compositions of the invention may be characterized by thefollowing structure (II):

wherein R′ is selected from the group consisting of H, CH₃,CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof;wherein R″ is selected from the group consisting of H,CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof;wherein x+y≦5; wherein y≧1; and wherein z=0 to 5.

A further preferred additional hueing dye may be characterized by thefollowing structure (III):

This dye is typically a mixture of compounds having an average of 3-10EO groups, preferably 5 EO groups per molecule.

Further additional shading dyes are those described in USPN 2008 34511A1 (Unilever). A preferred agent is “Solvent Violet 13”.

Suitable dye clay conjugates include dye clay conjugates selected fromthe group comprising at least one cationic/basic dye and a smectiteclay, and mixtures thereof. In another aspect, suitable dye clayconjugates include dye clay conjugates selected from the groupconsisting of one cationic/basic dye selected from the group consistingof C.I. Basic Yellow 1 through 108, C.I. Basic Orange 1 through 69, C.I.Basic Red 1 through 118, C.I. Basic Violet 1 through 51, C.I. Basic Blue1 through 164, C.I. Basic Green 1 through 14, C.I. Basic Brown 1 through23, CI Basic Black 1 through 11, and a clay selected from the groupconsisting of Montmorillonite clay, Hectorite clay, Saponite clay andmixtures thereof. In still another aspect, suitable dye clay conjugatesinclude dye clay conjugates selected from the group consisting of:Montmorillonite Basic Blue B7 C.I. 42595 conjugate, MontmorilloniteBasic Blue B9 C.I. 52015 conjugate, Montmorillonite Basic Violet V3 C.I.42555 conjugate, Montmorillonite Basic Green G1 C.I. 42040 conjugate,Montmorillonite Basic Red R1C.I. 45160 conjugate, Montmorillonite C.I.Basic Black 2 conjugate, Hectorite Basic Blue B7 C.I. 42595 conjugate,Hectorite Basic Blue B9 C.I. 52015 conjugate, Hectorite Basic Violet V3C.I. 42555 conjugate, Hectorite Basic Green G1 C.I. 42040 conjugate,Hectorite Basic Red R1 C.I. 45160 conjugate, Hectorite C.I. Basic Black2 conjugate, Saponite Basic Blue B7 C.I. 42595 conjugate, Saponite BasicBlue B9 C.I. 52015 conjugate, Saponite Basic Violet V3 C.I. 42555conjugate, Saponite Basic Green G1 C.I. 42040 conjugate, Saponite BasicRed R1 C.I. 45160 conjugate, Saponite C.I. Basic Black 2 conjugate andmixtures thereof.

Suitable pigments include pigments selected from the group consisting offlavanthrone, indanthrone, chlorinated indanthrone containing from 1 to4 chlorine atoms, pyranthrone, dichloropyranthrone,monobromodichloropyranthrone, dibromodichloropyranthrone,tetrabromopyranthrone, perylene-3,4,9,10-tetracarboxylic acid diimide,wherein the imide groups may be unsubstituted or substituted byC₁-C₃-alkyl or a phenyl or heterocyclic radical, and wherein the phenyland heterocyclic radicals may additionally carry substituents which donot confer solubility in water, anthrapyrimidinecarboxylic acid amides,violanthrone, isoviolanthrone, dioxazine pigments, copper phthalocyaninewhich may contain up to 2 chlorine atoms per molecule, polychloro-copperphthalocyanine or polybromochloro-copper phthalocyanine containing up to14 bromine atoms per molecule and mixtures thereof. Particularlypreferred are Pigment Blues 15 to 20, especially Pigment Blue 15 and/or16. Other suitable pigments include those selected from the groupconsisting of Ultramarine Blue (C.I. Pigment Blue 29), UltramarineViolet (C.I. Pigment Violet 15) and mixtures thereof. Suitable hueingagents are described in more detail in U.S. Pat. No. 7,208,459 B2.

Encapsulates.

The composition may comprise an encapsulate. In one aspect, anencapsulate comprising a core, a shell having an inner and outersurface, said shell encapsulating said core. The core may comprise anylaundry care adjunct, though typically the core may comprise materialselected from the group consisting of perfumes; brighteners; dyes;insect repellants; silicones; waxes; flavors; vitamins; fabric softeningagents; skin care agents in one aspect, paraffins; enzymes;anti-bacterial agents; bleaches; sensates; and mixtures thereof; andsaid shell may comprise a material selected from the group consisting ofpolyethylenes; polyamides; polyvinylalcohols, optionally containingother co-monomers; polystyrenes; polyisoprenes; polycarbonates;polyesters; polyacrylates; aminoplasts, in one aspect said aminoplastmay comprise a polyureas, polyurethane, and/or polyureaurethane, in oneaspect said polyurea may comprise polyoxymethyleneurea and/or melamineformaldehyde; polyolefins; polysaccharides, in one aspect saidpolysaccharide may comprise alginate and/or chitosan; gelatin; shellac;epoxy resins; vinyl polymers; water insoluble inorganics; silicone; andmixtures thereof. Preferred encapsulates comprise perfume. Preferredencapsulates comprise a shell which may comprise melamine formaldehydeand/or cross linked melamine formaldehyde. Preferred encapsulatescomprise a core material and a shell, said shell at least partiallysurrounding said core material, is disclosed. At least 75%, 85% or even90% of said encapsulates may have a fracture strength of from 0.2 MPa to10 MPa, and a benefit agent leakage of from 0% to 20%, or even less than10% or 5% based on total initial encapsulated benefit agent. Preferredare those in which at least 75%, 85% or even 90% of said encapsulatesmay have (i) a particle size of from 1 microns to 80 microns, 5 micronsto 60 microns, from 10 microns to 50 microns, or even from 15 microns to40 microns, and/or (ii) at least 75%, 85% or even 90% of saidencapsulates may have a particle wall thickness of from 30 nm to 250 nm,from 80 nm to 180 nm, or even from 100 nm to 160 nm. Formaldehydescavengers may be employed with the encapsulates, for example, in acapsule slurry and/or added to a composition before, during or after theencapsulates are added to such composition. Suitable capsules that canbe made by following the teaching of USPA 2008/0305982 A1; and/or USPA2009/0247449 A1. Alternatively, suitable capsules can be purchased fromAppleton Papers Inc. of Appleton, Wis. USA.

In a preferred aspect the composition may comprise a deposition aid,preferably in addition to encapsulates. Preferred deposition aids areselected from the group consisting of cationic and nonionic polymers.Suitable polymers include cationic starches, cationichydroxyethylcellulose, polyvinylformaldehyde, locust bean gum, mannans,xyloglucans, tamarind gum, polyethyleneterephthalate and polymerscontaining dimethylaminoethyl methacrylate, optionally with one or moremonomers selected from the group comprising acrylic acid and acrylamide.

Perfume.

Preferred compositions of the invention comprise perfume. Typically thecomposition comprises a perfume that comprises one or more perfume rawmaterials, selected from the group as described in WO08/87497. However,any perfume useful in a laundry care composition may be used. Apreferred method of incorporating perfume into the compositions of theinvention is via an encapsulated perfume particle comprising either awater-soluble hydroxylic compound or melamine-formaldehyde or modifiedpolyvinyl alcohol. In one aspect the encapsulate comprises (a) an atleast partially water-soluble solid matrix comprising one or morewater-soluble hydroxylic compounds, preferably starch; and (b) a perfumeoil encapsulated by the solid matrix. In a further aspect the perfumemay be pre-complexed with a polyamine, preferably a polyethylenimine soas to form a Schiff base.

Polymers.

The composition may comprise one or more polymers. Examples areoptionally modified carboxymethylcellulose, poly(vinyl-pyrrolidone),poly (ethylene glycol), poly(vinyl alcohol),poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates suchas polyacrylates, maleic/acrylic acid copolymers and laurylmethacrylate/acrylic acid co-polymers.

The composition may comprise one or more amphiphilic cleaning polymerssuch as the compound having the following general structure:bis((C₂H₅O)(C₂H₄O)n)(CH₃)—N⁺—C_(x)H_(2x)—N⁺—(CH₃)-bis((C₂H₅O)(C₂H₄O)n),wherein n=from 20 to 30, and x=from 3 to 8, or sulphated or sulphonatedvariants thereof. In one aspect, this polymer is sulphated orsulphonated to provide a zwitterionic soil suspension polymer.

The composition preferably comprises amphiphilic alkoxylated greasecleaning polymers which have balanced hydrophilic and hydrophobicproperties such that they remove grease particles from fabrics andsurfaces. Preferred amphiphilic alkoxylated grease cleaning polymerscomprise a core structure and a plurality of alkoxylate groups attachedto that core structure. These may comprise alkoxylatedpolyalkylenimines, preferably having an inner polyethylene oxide blockand an outer polypropylene oxide block. Typically these may beincorporated into the compositions of the invention in amounts of from0.005 to 10 wt %, generally from 0.5 to 8 wt %.

Alkoxylated polycarboxylates such as those prepared from polyacrylatesare useful herein to provide additional grease removal performance. Suchmaterials are described in WO 91/08281 and PCT 90/01815. Chemically,these materials comprise polyacrylates having one ethoxy side-chain perevery 7-8 acrylate units. The side-chains are of the formula—(CH₂CH₂O)_(m) (CH₂)_(n)CH₃ wherein m is 2-3 and n is 6-12. Theside-chains are ester-linked to the polyacrylate “backbone” to provide a“comb” polymer type structure. The molecular weight can vary, but istypically in the range of about 2000 to about 50,000. Such alkoxylatedpolycarboxylates can comprise from about 0.05% to about 10%, by weight,of the compositions herein.

Mixtures of cosurfactants and other adjunct ingredients, areparticularly suited to be used with an amphiphilic graft co-polymer.Preferred amphiphilic graft co-polymer(s) comprise (i) polyethyeleneglycol backbone; and (ii) and at least one pendant moiety selected frompolyvinyl acetate, polyvinyl alcohol and mixtures thereof. A preferredamphiphilic graft co-polymer is Sokalan HP22, supplied from BASF.Suitable polymers include random graft copolymers, preferably apolyvinyl acetate grafted polyethylene oxide copolymer having apolyethylene oxide backbone and multiple polyvinyl acetate side chains.The molecular weight of the polyethylene oxide backbone is preferablyabout 6000 and the weight ratio of the polyethylene oxide to polyvinylacetate is about 40 to 60 and no more than 1 grafting point per 50ethylene oxide units. Typically these are incorporated into thecompositions of the invention in amounts from 0.005 to 10 wt %, moreusually from 0.05 to 8 wt %. Preferably the composition comprises one ormore carboxylate polymer, such as a maleate/acrylate random copolymer orpolyacrylate homopolymer. In one aspect, the carboxylate polymer is apolyacrylate homopolymer having a molecular weight of from 4,000 Da to9,000 Da, or from 6,000 Da to 9,000 Da. Typically these are incorporatedinto the compositions of the invention in amounts from 0.005 to 10 wt %,or from 0.05 to 8 wt %.

Preferably the composition comprises one or more soil release polymers.Examples include soil release polymers having a structure as defined byone of the following Formulae (IV), (V) or (VI):—[(OCHR¹—CHR²)_(a)—O—OC—Ar—CO—]_(d)  (IV)—[(OCHR³—CHR⁴)_(b)—O—OC-sAr—CO—]_(e)  (V)—[(OCHR⁵—CHR⁶)_(c)—OR⁷]_(f)  (VI)

wherein:

a, b and c are from 1 to 200;

d, e and f are from 1 to 50;

Ar is a 1,4-substituted phenylene;

sAr is 1,3-substituted phenylene substituted in position 5 with SO₃Me;

Me is L1, K, Mg/2, Ca/2, A1/3, ammonium, mono-, di-, tri-, ortetraalkylammonium wherein the alkyl groups are C₁-C₁₈ alkyl or C₂-C₁₀hydroxyalkyl, or mixtures thereof;

R¹, R², R³, R⁴, R⁵ and R⁶ are independently selected from H or C₁-C₁₈ n-or iso-alkyl; and

R⁷ is a linear or branched C₁-C₁₈ alkyl, or a linear or branched C₂-C₃₀alkenyl, or a cycloalkyl group with 5 to 9 carbon atoms, or a C₈-C₃₀aryl group, or a C₆-C₃₀ arylalkyl group.

Suitable soil release polymers are polyester soil release polymers suchas Repel-o-tex polymers, including Repel-o-tex SF, SF-2 and SRP6supplied by Rhodia. Other suitable soil release polymers include Texcarepolymers, including Texcare SRA100, SRA300, SRN100, SRN170, SRN240,SRN300 and SRN325 supplied by Clariant. Other suitable soil releasepolymers are Marloquest polymers, such as Marloquest SL supplied bySasol.

Preferably the composition comprises one or more cellulosic polymer,including those selected from alkyl cellulose, alkyl alkoxyalkylcellulose, carboxyalkyl cellulose, alkyl carboxyalkyl cellulose.Preferred cellulosic polymers are selected from the group comprisingcarboxymethyl cellulose, methyl cellulose, methyl hydroxyethylcellulose, methyl carboxymethyl cellulose, and mixtures thereof. In oneaspect, the carboxymethyl cellulose has a degree of carboxymethylsubstitution from 0.5 to 0.9 and a molecular weight from 100,000 Da to300,000 Da.

Enzymes.

Preferably the composition comprises one or more enzymes. Preferredenzymes provide cleaning performance and/or fabric care benefits.Examples of suitable enzymes include, but are not limited to,hemicellulases, peroxidases, proteases, cellulases, xylanases, lipases,phospholipases, esterases, cutinases, pectinases, mannanases, pectatelyases, keratinases, reductases, oxidases, phenoloxidases,lipoxygenases, ligninases, pullulanases, tannases, pentosanases,malanases, β-glucanases, arabinosidases, hyaluronidase, chondroitinase,laccase, and amylases, or mixtures thereof. A typical combination is anenzyme cocktail that may comprise, for example, a protease and lipase inconjunction with amylase. When present in the composition, theaforementioned additional enzymes may be present at levels from about0.00001% to about 2%, from about 0.0001% to about 1% or even from about0.001% to about 0.5% enzyme protein by weight of the composition.

Proteases.

Preferably the composition comprises one or more proteases. Suitableproteases include metalloproteases and serine proteases, includingneutral or alkaline microbial serine proteases, such as subtilisins (EC3.4.21.62). Suitable proteases include those of animal, vegetable ormicrobial origin. In one aspect, such suitable protease may be ofmicrobial origin. The suitable proteases include chemically orgenetically modified mutants of the aforementioned suitable proteases.In one aspect, the suitable protease may be a serine protease, such asan alkaline microbial protease or/and a trypsin-type protease. Examplesof suitable neutral or alkaline proteases include:

(a) subtilisins (EC 3.4.21.62), including those derived from Bacillus,such as Bacillus lentus, B. alkalophilus, B. subtilis, B.amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii described inU.S. Pat. No. 6,312,936 B1, U.S. Pat. No. 5,679,630, U.S. Pat. No.4,760,025, U.S. Pat. No. 7,262,042 and WO09/021,867.

(b) trypsin-type or chymotrypsin-type proteases, such as trypsin (e.g.,of porcine or bovine origin), including the Fusarium protease describedin WO 89/06270 and the chymotrypsin proteases derived from Cellumonasdescribed in WO 05/052161 and WO 05/052146.

(c) metalloproteases, including those derived from Bacillusamyloliquefaciens described in WO 07/044,993A2.

Preferred proteases include those derived from Bacillus gibsonii orBacillus Lentus.

Suitable commercially available protease enzymes include those soldunder the trade names Alcalase®, Savinase®, Primase®, Durazym®,Polarzyme®, Kannase®, Liquanase®, Liquanase Ultra®, Savinase Ultra®,Ovozyme®, Neutrase®, Everlase® and Esperase® by Novozymes A/S (Denmark),those sold under the tradename Maxatase®, Maxacal®, Maxapem®,Properase®, Purafect®, Purafect Prime®, Purafect Ox®, FN3®, FN4®,Excellase® and Purafect OXP® by Genencor International, those sold underthe tradename Opticlean® and Optimase® by Solvay Enzymes, thoseavailable from Henkel/Kemira, namely BLAP (sequence shown in FIG. 29 ofU.S. Pat. No. 5,352,604 with the following mutationsS99D+S100R+S103A+V104I+G159S, hereinafter referred to as BLAP), BLAP R(BLAP with S3T+V4I+V199M+V205I+L217D), BLAP X (BLAP with S3T+V4I+V205I)and BLAP F49 (BLAP with S3T+V4I+A194P+V199M+V205I+L217D)—all fromHenkel/Kemira; and KAP (Bacillus alkalophilus subtilisin with mutationsA230V+S256G+S259N) from Kao.

Amylases.

Preferably the composition may comprise an amylase. Suitablealpha-amylases include those of bacterial or fungal origin. Chemicallyor genetically modified mutants (variants) are included. A preferredalkaline alpha-amylase is derived from a strain of Bacillus, such asBacillus licheniformis, Bacillus amyloliquefaciens, Bacillusstearothermophilus, Bacillus subtilis, or other Bacillus sp., such asBacillus sp. NCIB 12289, NCIB 12512, NCIB 12513, DSM 9375 (U.S. Pat. No.7,153,818) DSM 12368, DSMZ no. 12649, KSM AP1378 (WO 97/00324), KSM K36or KSM K38 (EP 1,022,334). Preferred amylases include:

(a) the variants described in WO 94/02597, WO 94/18314, WO96/23874 andWO 97/43424, especially the variants with substitutions in one or moreof the following positions versus the enzyme listed as SEQ ID No. 2 inWO 96/23874: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190,197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444.

(b) the variants described in U.S. Pat. No. 5,856,164 and WO99/23211, WO96/23873, WO00/60060 and WO 06/002643, especially the variants with oneor more substitutions in the following positions versus the AA560 enzymelisted as SEQ ID No. 12 in WO 06/002643:

26, 30, 33, 82, 37, 106, 118, 128, 133, 149, 150, 160, 178, 182, 186,193, 203, 214, 231, 256, 257, 258, 269, 270, 272, 283, 295, 296, 298,299, 303, 304, 305, 311, 314, 315, 318, 319, 339, 345, 361, 378, 383,419, 421, 437, 441, 444, 445, 446, 447, 450, 461, 471, 482, 484,preferably that also contain the deletions of D183* and G184*.

(c) variants exhibiting at least 90% identity with SEQ ID No. 4 inWO06/002643, the wild-type enzyme from Bacillus SP722, especiallyvariants with deletions in the 183 and 184 positions and variantsdescribed in WO 00/60060, which is incorporated herein by reference.

(d) variants exhibiting at least 95% identity with the wild-type enzymefrom Bacillus sp. 707 (SEQ ID NO:7 in U.S. Pat. No. 6,093,562),especially those comprising one or more of the following mutations M202,M208, S255, R172, and/or M261. Preferably said amylase comprises one ormore of M202L, M202V, M202S, M202T, M202I, M202Q, M202W, S255N and/orR172Q. Particularly preferred are those comprising the M202L or M202Tmutations.

(e) variants described in WO 09/149,130, preferably those exhibiting atleast 90% identity with SEQ ID NO: 1 or SEQ ID NO:2 in WO 09/149,130,the wild-type enzyme from Geobacillus Stearophermophilus or a truncatedversion thereof.

Suitable commercially available alpha-amylases include DURAMYL®,LIQUEZYME®, TERMAMYL®, TERMAMYL ULTRA®, NATALASE®, SUPRAMYL®,STAINZYME®, STAINZYME PLUS®, FUNGAMYL® and BAN® (Novozymes A/S,Bagsvaerd, Denmark), KEMZYM® AT 9000 Biozym Biotech Trading GmbHWehlistrasse 27b A-1200 Wien Austria, RAPIDASE®, PURASTAR®, ENZYSIZE®,OPTISIZE HT PLUS®, POWERASE® and PURASTAR OXAM® (Genencor InternationalInc., Palo Alto, Calif.) and KAM® (Kao, 14-10 Nihonbashi Kayabacho,1-chome, Chuo-ku Tokyo 103-8210, Japan). In one aspect, suitableamylases include NATALASE®, STAINZYME® and STAINZYME PLUS@ and mixturesthereof.

Lipases.

Preferably the invention comprises one or more lipases, including “firstcycle lipases” such as those described in U.S. Pat. No. 6,939,702 B1 andUS PA 2009/0217464. Preferred lipases are first-wash lipases. In oneembodiment of the invention the composition comprises a first washlipase. First wash lipases includes a lipase which is a polypeptidehaving an amino acid sequence which: (a) has at least 90% identity withthe wild-type lipase derived from Humicola lanuginosa strain DSM 4109;(b) compared to said wild-type lipase, comprises a substitution of anelectrically neutral or negatively charged amino acid at the surface ofthe three-dimensional structure within 15A of E1 or Q249 with apositively charged amino acid; and (c) comprises a peptide addition atthe C-terminal; and/or (d) comprises a peptide addition at theN-terminal and/or (e) meets the following limitations: i) comprises anegative amino acid in position E210 of said wild-type lipase; ii)comprises a negatively charged amino acid in the region corresponding topositions 90-101 of said wild-type lipase; and iii) comprises a neutralor negative amino acid at a position corresponding to N94 or saidwild-type lipase and/or has a negative or neutral net electric charge inthe region corresponding to positions 90-101 of said wild-type lipase.Preferred arevariants of the wild-type lipase from Thermomyceslanuginosus comprising one or more of the T231R and N233R mutations. Thewild-type sequence is the 269 amino acids (amino acids 23-291) of theSwissprot accession number Swiss-Prot 059952 (derived from Thermomyceslanuginosus (Humicola lanuginosa)). Preferred lipases would includethose sold under the tradenames Lipex® and Lipolex® and Lipoclean®.

Endoglucanases.

Other preferred enzymes include microbial-derived endoglucanasesexhibiting endo-beta-1,4-glucanase activity (E.C. 3.2.1.4), including abacterial polypeptide endogenous to a member of the genus Bacillus whichhas a sequence of at least 90%, 94%, 97% and even 99% identity to theamino acid sequence SEQ ID NO:2 in U.S. Pat. No. 7,141,403B2) andmixtures thereof. Suitable endoglucanases are sold under the tradenamesCelluclean® and Whitezyme® (Novozymes A/S, Bagsvaerd, Denmark).

Pectate Lyases.

Other preferred enzymes include pectate lyases sold under the tradenamesPectawash®, Pectaway®, Xpect® and mannanases sold under the tradenamesMannaway® (all from Novozymes A/S, Bagsvaerd, Denmark), and Purabrite®(Genencor International Inc., Palo Alto, Calif.).

Bleaching Agents.

It may be preferred for the composition to comprise one or morebleaching agents. Suitable bleaching agents other than bleachingcatalysts include photobleaches, bleach activators, hydrogen peroxide,sources of hydrogen peroxide, pre-formed peracids and mixtures thereof.In general, when a bleaching agent is used, the compositions of thepresent invention may comprise from about 0.1% to about 50% or even fromabout 0.1% to about 25% bleaching agent or mixtures of bleaching agentsby weight of the subject composition. Examples of suitable bleachingagents include:

(1) photobleaches for example sulfonated zinc phthalocyanine sulfonatedaluminium phthalocyanines, xanthene dyes and mixtures thereof;

(2) pre-formed peracids: Suitable preformed peracids include, but arenot limited to compounds selected from the group consisting ofpre-formed peroxyacids or salts thereof typically a percarboxylic acidsand salts, percarbonic acids and salts, perimidic acids and salts,peroxymonosulfuric acids and salts, for example, Oxone®, and mixturesthereof. Suitable examples include peroxycarboxylic acids or saltsthereof, or peroxysulphonic acids or salts thereof. Typicalperoxycarboxylic acid salts suitable for use herein have a chemicalstructure corresponding to the following chemical formula:

wherein: R¹⁴ is selected from alkyl, aralkyl, cycloalkyl, aryl orheterocyclic groups; the R¹⁴ group can be linear or branched,substituted or unsubstituted; having, when the peracid is hydrophobic,from 6 to 14 carbon atoms, or from 8 to 12 carbon atoms and, when theperacid is hydrophilic, less than 6 carbon atoms or even less than 4carbon atoms and Y is any suitable counter-ion that achieves electriccharge neutrality, preferably Y is selected from hydrogen, sodium orpotassium. Preferably, R¹⁴ is a linear or branched, substituted orunsubstituted C₆₋₉ alkyl. Preferably, the peroxyacid or salt thereof isselected from peroxyhexanoic acid, peroxyheptanoic acid, peroxyoctanoicacid, peroxynonanoic acid, peroxydecanoic acid, any salt thereof, or anycombination thereof. Particularly preferred peroxyacids arephthalimido-peroxy-alkanoic acids, in particular ε-phthalimido peroxyhexanoic acid (PAP). Preferably, the peroxyacid or salt thereof has amelting point in the range of from 30° C. to 60° C.

The pre-formed peroxyacid or salt thereof can also be a peroxysulphonicacid or salt thereof, typically having a chemical structurecorresponding to the following chemical formula:

wherein: R¹⁵ is selected from alkyl, aralkyl, cycloalkyl, aryl orheterocyclic groups; the R¹⁵ group can be linear or branched,substituted or unsubstituted; and Z is any suitable counter-ion thatachieves electric charge neutrality, preferably Z is selected fromhydrogen, sodium or potassium. Preferably R¹⁵ is a linear or branched,substituted or unsubstituted C₄₋₁₄, preferably C₆₋₁₄ alkyl. Preferablysuch bleach components may be present in the compositions of theinvention in an amount from 0.01 to 50%, most preferably from 0.1% to20%.

(3) sources of hydrogen peroxide, for example, inorganic perhydratesalts, including alkali metal salts such as sodium salts of perborate(usually mono- or tetra-hydrate), percarbonate, persulphate,perphosphate, persilicate salts and mixtures thereof. In one aspect ofthe invention the inorganic perhydrate salts are selected from the groupconsisting of sodium salts of perborate, percarbonate and mixturesthereof. When employed, inorganic perhydrate salts are typically presentin amounts of from 0.05 to 40 wt %, or 1 to 30 wt % of the overallcleaning and/or treatment composition, such as fabric and home careproduct, and are typically incorporated into such cleaning and/ortreatment composition, such as fabric and home care products, as acrystalline solid that may be coated. Suitable coatings include,inorganic salts such as alkali metal silicate, carbonate or borate saltsor mixtures thereof, or organic materials such as water-soluble ordispersible polymers, waxes, oils or fatty soaps; and(4) bleach activators having R—(C═O)-L wherein R is an alkyl group,optionally branched, having, when the bleach activator is hydrophobic,from 6 to 14 carbon atoms, or from 8 to 12 carbon atoms and, when thebleach activator is hydrophilic, less than 6 carbon atoms or even lessthan 4 carbon atoms; and L is leaving group. Examples of suitableleaving groups are benzoic acid and derivatives thereof—especiallybenzene sulphonate. Suitable bleach activators include dodecanoyloxybenzene sulphonate, decanoyl oxybenzene sulphonate, decanoyloxybenzoic acid or salts thereof, 3,5,5-trimethyl hexanoyloxybenzenesulphonate, tetraacetyl ethylene diamine (TAED) and nonanoyloxybenzenesulphonate (NOBS). Suitable bleach activators are also disclosed in WO98/17767. While any suitable bleach activator may be employed, in oneaspect of the invention the subject composition may comprise NOBS, TAEDor mixtures thereof.(5) Bleach Catalysts. The compositions of the present invention may alsoinclude one or more bleach catalysts capable of accepting an oxygen atomfrom a peroxyacid and/or salt thereof, and transferring the oxygen atomto an oxidizeable substrate. Suitable bleach catalysts include, but arenot limited to: iminium cations and polyions; iminium zwitterions;modified amines; modified amine oxides; N-sulphonyl imines; N-phosphonylimines; N-acyl imines; thiadiazole dioxides; perfluoroimines; cyclicsugar ketones and alpha amino-ketones and mixtures thereof. Suitablealpha amino ketones are for example as described in WO 2012/000846 A1,WO 2008/015443 A1, and WO 2008/014965 A1. Suitable mixtures are asdescribed in USPA 2007/0173430 A1.

Without wishing to be bound by theory, the inventors believe thatcontrolling the electophilicity and hydrophobicity in this abovedescribed manner enables the bleach ingredient to be deliveredsubstantially only to areas of the fabric that are more hydrophobic, andthat contain electron rich soils, including visible chromophores, thatare susceptible to bleaching by highly electrophilic oxidants.

In one aspect, the bleach catalyst has a structure corresponding togeneral formula below:

wherein R¹³ is selected from the group consisting of 2-ethylhexyl,2-propylheptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, n-dodecyl,n-tetradecyl, n-hexadecyl, n-octadecyl, iso-nonyl, iso-decyl,iso-tridecyl and iso-pentadecyl;

(6) The composition may preferably comprise catalytic metal complexes.One preferred type of metal-containing bleach catalyst is a catalystsystem comprising a transition metal cation of defined bleach catalyticactivity, such as copper, iron, titanium, ruthenium, tungsten,molybdenum, or manganese cations, an auxiliary metal cation havinglittle or no bleach catalytic activity, such as zinc or aluminumcations, and a sequestrate having defined stability constants for thecatalytic and auxiliary metal cations, particularlyethylenediaminetetraacetic acid,ethylenediaminetetra(methylenephosphonic acid) and water-soluble saltsthereof. Such catalysts are disclosed in U.S. Pat. No. 4,430,243.

If desired, the compositions herein can be catalyzed by means of amanganese compound. Such compounds and levels of use are well known inthe art and include, for example, the manganese-based catalystsdisclosed in U.S. Pat. No. 5,576,282.

Cobalt bleach catalysts useful herein are known, and are described, forexample, in U.S. Pat. No. 5,597,936; U.S. Pat. No. 5,595,967. Suchcobalt catalysts are readily prepared by known procedures, such astaught for example in U.S. Pat. No. 5,597,936, and U.S. Pat. No.5,595,967.

Compositions herein may also suitably include a transition metal complexof ligands such as bispidones (WO 05/042532 A1) and/or macropolycyclicrigid ligands—abbreviated as “MRLs”. As a practical matter, and not byway of limitation, the compositions and processes herein can be adjustedto provide on the order of at least one part per hundred million of theactive MRL species in the aqueous washing medium, and will typicallyprovide from about 0.005 ppm to about 25 ppm, from about 0.05 ppm toabout 10 ppm, or even from about 0.1 ppm to about 5 ppm, of the MRL inthe wash liquor.

Suitable transition-metals in the instant transition-metal bleachcatalyst include, for example, manganese, iron and chromium. SuitableMRLs include 5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane.

Suitable transition metal MRLs are readily prepared by known procedures,such as taught for example in WO 00/32601, and U.S. Pat. No. 6,225,464.

When present, the source of hydrogen peroxide/peracid and/or bleachactivator is generally present in the composition in an amount of fromabout 0.1 to about 60 wt %, from about 0.5 to about 40 wt % or even fromabout 0.6 to about 10 wt % based on the cleaning and/or treatmentcomposition, such as fabric and home care product. One or morehydrophobic peracids or precursors thereof may be used in combinationwith one or more hydrophilic peracid or precursor thereof.

Typically hydrogen peroxide source and bleach activator will beincorporated together. The amounts of hydrogen peroxide source andperacid or bleach activator may be selected such that the molar ratio ofavailable oxygen (from the peroxide source) to peracid is from 1:1 to35:1, or even 2:1 to 10:1.

Surfactant.

Preferably the composition comprises a surfactant or surfactant system.The surfactant can be selected from nonionic, anionic, cationic,amphoteric, ampholytic, amphiphilic, zwitterionic, semi-polar nonionicsurfactants and mixtures thereof. Preferred compositions comprise amixture of surfactants/surfactant system. Preferred surfactant systemscomprise one or more anionic surfactants, most preferably in combinationwith a co-surfactant, most preferably a nonionic and/or amphotericand/or zwitterionic surfactant. Preferred surfactant systems compriseboth anionic and nonionic surfactant, preferably in weight ratios from90:1 to 1:90. In some instances a weight ratio of anionic to nonionicsurfactant of at least 1:1 is preferred. However a ratio below 10:1 maybe preferred. When present, the total surfactant level is preferablyfrom 0.1% to 60%, from 1% to 50% or even from 5% to 40% by weight of thesubject composition.

Preferably the composition comprises an anionic detersive surfactant,preferably sulphate and/or sulphonate surfactants. Preferred examplesinclude alkyl benzene sulphonates, alkyl sulphates and alkyl alkoxylatedsulphates. Preferred sulphonates are C₁₀₋₁₃ alkyl benzene sulphonate.Suitable alkyl benzene sulphonate (LAS) may be obtained, by sulphonatingcommercially available linear alkyl benzene (LAB); suitable LAB includeslow 2-phenyl LAB, such as those supplied by Sasol under the tradenameIsochem® or those supplied by Petresa under the tradename Petrelab®,other suitable LAB include high 2-phenyl LAB, such as those supplied bySasol under the tradename Hyblene®. A suitable anionic detersivesurfactant is alkyl benzene sulphonate that is obtained by DETALcatalyzed process, although other synthesis routes, such as HF, may alsobe suitable. In one aspect a magnesium salt of LAS is used.

Preferred sulphate detersive surfactants include alkyl sulphate,typically C₈₋₃₈ alkyl sulphate, or predominantly C₃₋₂ alkyl sulphate. Afurther preferred alkyl sulphate is alkyl alkoxylated sulphate,preferably a C₈₋₁₈ alkyl alkoxylated sulphate. Preferably thealkoxylating group is an ethoxylating group. Typically the alkylalkoxylated sulphate has an average degree of alkoxylation of from 0.5to 30 or 20, or from 0.5 to 10. Particularly preferred are C₈₋₁₈ alkylethoxylated sulphate having an average degree of ethoxylation of from0.5 to 10, from 0.5 to 7, from 0.5 to 5 or even from 0.5 to 3.

The alkyl sulphate, alkyl alkoxylated sulphate and alkyl benzenesulphonates may be linear or branched, substituted or un-substituted.When the surfactant is branched, preferably the surfactant will comprisea mid-chain branched sulphate or sulphonate surfactant. Preferably thebranching groups comprise C₁₋₄ alkyl groups, typically methyl and/orethyl groups.

Preferably the composition comprises a nonionic detersive surfactant.Suitable non-ionic surfactants are selected from the group consistingof: C₈-C₁₈ alkyl ethoxylates, such as, NEODOL® non-ionic surfactantsfrom Shell; C₆-C₁, alkyl phenol alkoxylates wherein the alkoxylate unitsmay be ethyleneoxy units, propyleneoxy units or a mixture thereof;C₁₂-C₁₈ alcohol and C₆-C₁₂ alkyl phenol condensates with ethyleneoxide/propylene oxide block polymers such as Pluronic® from BASF;C₁₄-C₂₂ mid-chain branched alcohols; C₁₄-C₂₂ mid-chain branched alkylalkoxylates, typically having an average degree of alkoxylation of from1 to 30; alkylpolysaccharides, in one aspect, alkylpolyglycosides;polyhydroxy fatty acid amides; ether capped poly(oxyalkylated) alcoholsurfactants; and mixtures thereof.

Suitable non-ionic detersive surfactants include alkyl polyglucosideand/or an alkyl alkoxylated alcohol.

In one aspect, non-ionic detersive surfactants include alkyl alkoxylatedalcohols, in one aspect C₈₋₁₈ alkyl alkoxylated alcohol, for example aC₈₋₁₈ alkyl ethoxylated alcohol, the alkyl alkoxylated alcohol may havean average degree of alkoxylation of from 1 to 80, preferably from 1 to50, most preferably from 1 to 30, from 1 to 20, or from 1 to 10. In oneaspect, the alkyl alkoxylated alcohol may be a C₈₋₁₈ alkyl ethoxylatedalcohol having an average degree of ethoxylation of from 1 to 10, from 1to 7, more from 1 to 5 or from 3 to 7, or even below 3 or 2.

The alkyl alkoxylated alcohol can be linear or branched, and substitutedor un-substituted.

Suitable nonionic surfactants include those with the tradename Lutensol®from BASF.

Suitable cationic detersive surfactants include alkyl pyridiniumcompounds, alkyl quaternary ammonium compounds, alkyl quaternaryphosphonium compounds, alkyl ternary sulphonium compounds, and mixturesthereof.

Suitable cationic detersive surfactants are quaternary ammoniumcompounds having the general formula:(R)(R₁)(R₂)(R₃)N⁺X⁻

wherein, R is a linear or branched, substituted or unsubstituted C₆₋₁₈alkyl or alkenyl moiety, R₁ and R₂ are independently selected frommethyl or ethyl moieties, R₃ is a hydroxyl, hydroxymethyl or ahydroxyethyl moiety, X is an anion which provides charge neutrality,suitable anions include: halides, for example chloride; sulphate; andsulphonate. Suitable cationic detersive surfactants are mono-C₆₋₁₈ alkylmono-hydroxyethyl di-methyl quaternary ammonium chlorides. Highlysuitable cationic detersive surfactants are mono-C₈₋₃₀ alkylmono-hydroxyethyl di-methyl quaternary ammonium chloride, mono-C₁₀₋₁₂alkyl mono-hydroxyethyl di-methyl quaternary ammonium chloride andmono-C₁₀ alkyl mono-hydroxyethyl di-methyl quaternary ammonium chloride.

Suitable amphoteric/zwitterionic surfactants include amine oxides andbetaines.

Amine-neutralized anionic surfactants—Anionic surfactants of the presentinvention and adjunct anionic cosurfactants, may exist in an acid form,and said acid form may be neutralized to form a surfactant salt which isdesirable for use in the present detergent compositions. Typical agentsfor neutralization include the metal counterion base such as hydroxides,eg, NaOH or KOH. Further preferred agents for neutralizing anionicsurfactants of the present invention and adjunct anionic surfactants orcosurfactants in their acid forms include ammonia, amines, oralkanolamines. Alkanolamines are preferred. Suitable non-limitingexamples including monoethanolamine, diethanolamine, triethanolamine,and other linear or branched alkanolamines known in the art; forexample, highly preferred alkanolamines include 2-amino-1-propanol,1-aminopropanol, monoisopropanolamine, or 1-amino-3-propanol. Amineneutralization may be done to a full or partial extent, e.g. part of theanionic surfactant mix may be neutralized with sodium or potassium andpart of the anionic surfactant mix may be neutralized with amines oralkanolamines.

Builders.

Preferably the composition comprises one or more builders or a buildersystem. When a builder is used, the composition of the invention willtypically comprise at least 1%, from 2% to 60% builder. It may bepreferred that the composition comprises low levels of phosphate saltand/or zeolite, for example from 1 to 10 or 5 wt %. The composition mayeven be substantially free of strong builder; substantially free ofstrong builder means “no deliberately added” zeolite and/or phosphate.Typical zeolite builders include zeolite A, zeolite P and zeolite MAP. Atypical phosphate builder is sodium tripolyphosphate.

Chelating Agent.

Preferably the composition comprises chelating agents and/or crystalgrowth inhibitor. Suitable molecules include copper, iron and/ormanganese chelating agents and mixtures thereof. Suitable moleculesinclude aminocarboxylates, aminophosphonates, succinates, salts thereof,and mixtures thereof. Non-limiting examples of suitable chelants for useherein include ethylenediaminetetracetates,N-(hydroxyethyl)ethylenediaminetriacetates, nitrilotriacetates,ethylenediamine tetraproprionates, triethylenetetraaminehexacetates,diethylenetriamine-pentaacetates, ethanoldiglycines,ethylenediaminetetrakis (methylenephosphonates), diethylenetriaminepenta(methylene phosphoric acid) (DTPMP), ethylenediamine disuccinate(EDDS), hydroxyetlianedimethylenephosphonic acid (HEDP),methylglycinediacetic acid (MGDA), diethylenetriaminepentaacetic acid(DTPA), salts thereof, and mixtures thereof. Other nonlimiting examplesof chelants of use in the present invention are found in U.S. Pat. Nos.7,445,644, 7,585,376 and 2009/0176684A1. Other suitable chelating agentsfor use herein are the commercial DEQUEST series, and chelants fromMonsanto, DuPont, and Nalco, Inc.

Dye Transfer Inhibitor (DTI).

The composition may comprise one or more dye transfer inhibiting agents.In one embodiment of the invention the inventors have surprisingly foundthat compositions comprising polymeric dye transfer inhibiting agents inaddition to the specified dye give improved performance. This issurprising because these polymers prevent dye deposition. Suitable dyetransfer inhibitors include, but are not limited to,polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers ofN-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones andpolyvinylimidazoles or mixtures thereof. Suitable examples includePVP-K15, PVP-K30, ChromaBond S-400, ChromaBond S-403E and ChromabondS-100 from Ashland Aqualon, and Sokalan HP165, Sokalan HP50, SokalanHP53, Sokalan HP59, Sokalan® HP 56K, Sokalan® HP 66 from BASF. Othersuitable DTIs are as described in WO2012/004134. When present in asubject composition, the dye transfer inhibiting agents may be presentat levels from about 0.0001% to about 10%, from about 0.01% to about 5%or even from about 0.1% to about 3% by weight of the composition.

Fluorescent Brightener.

Preferably the composition comprises one or more fluorescent brightener.Commercial optical brighteners which may be useful in the presentinvention can be classified into subgroups, which include, but are notlimited to, derivatives of stilbene, pyrazoline, coumarin, carboxylicacid, methinecyanines, dibenzothiophene-5,5-dioxide, azoles, 5- and6-membered-ring heterocycles, and other miscellaneous agents.Particularly preferred brighteners are selected from: sodium 2(4-styryl-3-sulfophenyl)-2H-napthol[1,2-d]triazole, disodium4,4′-bis{[(4-anilino-6-(N methyl-N-2 hydroxyethyl)amino1,3,5-triazin-2-yl)]amino}stilbene-2-{2-disulfonate, disodium4,4′-bis{[(4-anilino-6-morpholino-1,3,5-triazin-2-yl)]amino}stilbene-2-2′disulfonate,and disodium 4,4′-bis(2-sulfostyryl)biphenyl. Other examples of suchbrighteners are disclosed in “The Production and Application ofFluorescent Brightening Agents”, M. Zahradnik, Published by John Wiley &Sons, New York (1982). Specific nonlimiting examples of opticalbrighteners which are useful in the present compositions are thoseidentified in U.S. Pat. No. 4,790,856 and U.S. Pat. No. 3,646,015.

A preferred brightener has the structure below:

Suitable fluorescent brightener levels include lower levels of fromabout 0.01, from about 0.05, from about 0.1 or even from about 0.2 wt %to upper levels of 0.5 or even 0.75 wt %.

In one aspect the brightener may be loaded onto a clay to form aparticle.

Preferred brighteners are totally or predominantly (typically at least50 wt %, at least 75 wt %, at least 90 wt %, at least 99 wt %), inalpha-crystalline form. A highly preferred brightener comprises C.I.fluorescent brightener 260, preferably having the following structure:

This can be particularly useful as it dissolves well in cold water, forexample below 30 or 25 or even 20° C.

Preferably brighteners are incorporated in the composition in micronizedparticulate form, most preferably having a weight average primaryparticle size of from 3 to 30 micrometers, from 3 micrometers to 20micrometers, or from 3 to 10 micrometers.

The composition may comprise C.I. fluorescent brightener 260 inbeta-crystalline form, and the weight ratio of: (i) C.I. fluorescentbrightener 260 in alpha-crystalline form, to (ii) C.I. fluorescentbrightener 260 in beta-crystalline form may be at least 0.1, or at least0.6.

BE680847 relates to a process for making C.I fluorescent brightener 260in alpha-crystalline form.

Silicate Salts.

The composition may preferably also contain silicate salts, such assodium or potassium silicate. The composition may comprise from 0 wt %to less than 10 wt % silicate salt, to 9 wt %, or to 8 wt %, or to 7 wt%, or to 6 wt %, or to 5 wt %, or to 4 wt %, or to 3 wt %, or even to 2wt %, and preferably from above 0 wt %, or from 0.5 wt %, or even from 1wt % silicate salt. A suitable silicate salt is sodium silicate.

Dispersants.

The composition may preferably also contain dispersants. Suitablewater-soluble organic materials include the homo- or co-polymeric acidsor their salts, in which the polycarboxylic acid comprises at least twocarboxyl radicals separated from each other by not more than two carbonatoms.

Enzyme Stabilisers.

The composition may preferably comprise enzyme stabilizers. Anyconventional enzyme stabilizer may be used, for example by the presenceof water-soluble sources of calcium and/or magnesium ions in thefinished fabric and home care products that provide such ions to theenzymes. In case of aqueous compositions comprising protease, areversible protease inhibitor, such as a boron compound includingborate, or preferably 4-formyl phenylboronic acid, phenylboronic acidand derivatives thereof, or compounds such as calcium formate, sodiumformate and 1,2-propane diol can be added to further improve stability.

Solvent System.

The solvent system in the present compositions can be a solvent systemcontaining water alone or mixtures of organic solvents either without orpreferably with water. Preferred organic solvents include1,2-propanediol, ethanol, glycerol, dipropylene glycol, methyl propanediol and mixtures thereof. Other lower alcohols, C1-C4 alkanolaminessuch as monoethanolamine and triethanolamine, can also be used. Solventsystems can be absent, for example from anhydrous solid embodiments ofthe invention, but more typically are present at levels in the range offrom about 0.1% to about 98%, preferably at least about 1% to about 50%,more usually from about 5% to about 25%.

In some embodiments of the invention, the composition is in the form ofa structured liquid. Such structured liquids can either be internallystructured, whereby the structure is formed by primary ingredients (e.g.surfactant material) and/or externally structured by providing a threedimensional matrix structure using secondary ingredients (e.g. polymers,clay and/or silicate material), for use e.g. as thickeners. Thecomposition may comprise a structurant, preferably from 0.01 wt % to 5wt %, from 0.1 wt % to 2.0 wt % structurant. Examples of suitablestructurants are given in US2006/0205631A1, US2005/0203213A1, U.S. Pat.Nos. 7,294,611, 6,855,680. The structurant is typically selected fromthe group consisting of diglycerides and triglycerides, ethylene glycoldistearate, microcrystalline cellulose, cellulose-based materials,microfiber cellulose, hydrophobically modified alkali-swellableemulsions such as Polygel W30 (3VSigma), biopolymers, xanthan gum,gellan gum, hydrogenated castor oil, derivatives of hydrogenated castoroil such as non-ethoxylated derivatives thereof and mixtures thereof, inparticular, those selected from the group of hydrogenated castor oil,derivatives of hydrogenated castor oil, microfibullar cellulose,hydroxyfunctional crystalline materials, long chain fatty alcohols,12-hydroxystearic acids, clays and mixtures thereof. A preferredstructurant is described in. U.S. Pat. No. 6,855,680 which definessuitable hydroxyfunctional crystalline materials in detail. Preferred ishydrogenated castor oil. Non-limiting examples of useful structurantsinclude. Such structurants have a thread-like structuring system havinga range of aspect ratios. Other suitable structurants and the processesfor making them are described in WO2010/034736.

The composition of the present invention may comprise a high meltingpoint fatty compound. The high melting point fatty compound usefulherein has a melting point of 25° C. or higher, and is selected from thegroup consisting of fatty alcohols, fatty acids, fatty alcoholderivatives, fatty acid derivatives, and mixtures thereof. Suchcompounds of low melting point are not intended to be included in thissection. Non-limiting examples of the high melting point compounds arefound in International Cosmetic Ingredient Dictionary, Fifth Edition,1993, and CTFA Cosmetic Ingredient Handbook, Second Edition, 1992. Whenpresent, the high melting point fatty compound is preferably included inthe composition at a level of from 0.1% to 40%, preferably from 1% to30%, more preferably from 1.5% to 16% by weight of the composition, from1.5% to 8% in view of providing improved conditioning benefits such asslippery feel during the application to wet hair, softness andmoisturized feel on dry hair.

Cationic Polymer.

The compositions of the present invention may contain a cationicpolymer. Concentrations of the cationic polymer in the compositiontypically range from 0.05% to 3%, in another embodiment from 0.075% to2.0%, and in yet another embodiment from 0.1% to 1.0%. Suitable cationicpolymers will have cationic charge densities of at least 0.5 meq/gm, inanother embodiment at least 0.9 meq/gm, in another embodiment at least1.2 meq/gm, in yet another embodiment at least 1.5 meq/gm, but in oneembodiment also less than 7 meq/gm, and in another embodiment less than5 meq/gm, at the pH of intended use of the composition, which pH willgenerally range from pH 3 to pH 9, in one embodiment between pH 4 and pH8. Herein, “cationic charge density” of a polymer refers to the ratio ofthe number of positive charges on the polymer to the molecular weight ofthe polymer. The average molecular weight of such suitable cationicpolymers will generally be between 10,000 and 10 million, in oneembodiment between 50,000 and 5 million, and in another embodimentbetween 100,000 and 3 million.

Suitable cationic polymers for use in the compositions of the presentinvention contain cationic nitrogen-containing moieties such asquaternary ammonium or cationic protonated amino moieties. Any anioniccounterions can be used in association with the cationic polymers solong as the polymers remain soluble in water, in the composition, or ina coacervate phase of the composition, and so long as the counterionsare physically and chemically compatible with the essential componentsof the composition or do not otherwise unduly impair productperformance, stability or aesthetics. Nonlimiting examples of suchcounterions include halides (e.g., chloride, fluoride, bromide, iodide),sulfate and methylsulfate.

Nonlimiting examples of such polymers are described in the CTFA CosmeticIngredient Dictionary, 3rd edition, edited by Estrin, Crosley, andHaynes, (The Cosmetic, Toiletry, and Fragrance Association, Inc.,Washington, D.C. (1982)).

Other suitable cationic polymers for use in the composition includepolysaccharide polymers, cationic guar gum derivatives, quaternarynitrogen-containing cellulose ethers, synthetic polymers, copolymers ofetherified cellulose, guar and starch. When used, the cationic polymersherein are either soluble in the composition or are soluble in a complexcoacervate phase in the composition formed by the cationic polymer andthe anionic, amphoteric and/or zwitterionic surfactant componentdescribed hereinbefore. Complex coacervates of the cationic polymer canalso be formed with other charged materials in the composition.

Suitable cationic polymers are described in U.S. Pat. Nos. 3,962,418;3,958,581; and U.S. Publication No. 2007/0207109A1.

Nonionic Polymer.

The composition of the present invention may include a nonionic polymeras a conditioning agent. Polyalkylene glycols having a molecular weightof more than 1000 are useful herein. Useful are those having thefollowing general formula:

wherein R95 is selected from the group consisting of H, methyl, andmixtures thereof. Conditioning agents, and in particular silicones, maybe included in the composition. The conditioning agents useful in thecompositions of the present invention typically comprise a waterinsoluble, water dispersible, non-volatile, liquid that formsemulsified, liquid particles. Suitable conditioning agents for use inthe composition are those conditioning agents characterized generally assilicones (e.g., silicone oils, cationic silicones, silicone gums, highrefractive silicones, and silicone resins), organic conditioning oils(e.g., hydrocarbon oils, polyolefins, and fatty esters) or combinationsthereof, or those conditioning agents which otherwise form liquid,dispersed particles in the aqueous surfactant matrix herein. Suchconditioning agents should be physically and chemically compatible withthe essential components of the composition, and should not otherwiseunduly impair product stability, aesthetics or performance.

The concentration of the conditioning agent in the composition should besufficient to provide the desired conditioning benefits. Suchconcentration can vary with the conditioning agent, the conditioningperformance desired, the average size of the conditioning agentparticles, the type and concentration of other components, and otherlike factors.

The concentration of the silicone conditioning agent typically rangesfrom about 0.01% to about 10%. Non-limiting examples of suitablesilicone conditioning agents, and optional suspending agents for thesilicone, are described in U.S. Reissue Pat. No. 34,584, U.S. Pat. Nos.5,104,646; 5,106,609; 4,152,416; 2,826,551; 3,964,500; 4,364,837;6,607,717; 6,482,969; 5,807,956; 5,981,681; 6,207,782; 7,465,439;7,041,767; 7,217,777; US Patent Application Nos. 2007/0286837A1;2005/0048549A1; 2007/0041929A1; British Pat. No. 849,433; German PatentNo. DE 10036533, which are all incorporated herein by reference;Chemistry and Technology of Silicones, New York: Academic Press (1968);General Electric Silicone Rubber Product Data Sheets SE 30, SE 33, SE 54and SE 76; Silicon Compounds, Petrarch Systems, Inc. (1984); and inEncyclopedia of Polymer Science and Engineering, vol. 15, 2d ed., pp204-308, John Wiley & Sons, Inc. (1989).

Organic Conditioning Oil.

The compositions of the present invention may also comprise from about0.05% to about 3% of at least one organic conditioning oil as theconditioning agent, either alone or in combination with otherconditioning agents, such as the silicones (described herein). Suitableconditioning oils include hydrocarbon oils, polyolefins, and fattyesters. Also suitable for use in the compositions herein are theconditioning agents described by the Procter & Gamble Company in U.S.Pat. Nos. 5,674,478, and 5,750,122. Also suitable for use herein arethose conditioning agents described in U.S. Pat. Nos. 4,529,586,4,507,280, 4,663,158, 4,197,865, 4,217, 914, 4,381,919, and 4,422, 853.

Hygiene Agent.

The compositions of the present invention may also comprise componentsto deliver hygiene and/or malodour benefits such as one or more of zincricinoleate, thymol, quaternary ammonium salts such as Bardac®,polyethylenimines (such as Lupasol® from BASF) and zinc complexesthereof, silver and silver compounds, especially those designed toslowly release Ag+ or nano-silver dispersions.

Probiotics.

The composition may comprise probiotics, such as those described inWO2009/043709.

Suds Boosters.

The composition may preferably comprise suds boosters if high sudsing isdesired. Suitable examples are the C10-C16 alkanolamides or C10-C14alkyl sulphates, which are preferably incorporated at 1%-10% levels. TheC10-C14 monoethanol and diethanol amides illustrate a typical class ofsuch suds boosters. Use of such suds boosters with high sudsing adjunctsurfactants such as the amine oxides, betaines and sultaines noted aboveis also advantageous. If desired, water-soluble magnesium and/or calciumsalts such as MgCl2, MgSO4, CaCl2, CaSO4 and the like, can be added atlevels of, typically, 0.1%-2%, to provide additional suds and to enhancegrease removal performance.

Suds Supressor.

Compounds for reducing or suppressing the formation of suds may beincorporated into the compositions of the present invention. Sudssuppression can be of particular importance in the so-called “highconcentration cleaning process” as described in U.S. Pat. Nos. 4,489,455and 4,489,574, and in front-loading-style washing machines. A widevariety of materials may be used as suds suppressors, and sudssuppressors are well known to those skilled in the art. See, forexample, Kirk Othmer Encyclopedia of Chemical Technology, Third Edition,Volume 7, pages 430-447 (John Wiley & Sons, Inc., 1979). Examples ofsuds supressors include monocarboxylic fatty acid and soluble saltstherein, high molecular weight hydrocarbons such as paraffin, fatty acidesters (e.g., fatty acid triglycerides), fatty acid esters of monovalentalcohols, aliphatic C18-C40 ketones (e.g., stearone), N-alkylated aminotriazines, waxy hydrocarbons preferably having a melting point belowabout 100° C., silicone suds suppressors, and secondary alcohols. Sudssupressors are described in U.S. Pat. Nos. 2,954,347; 4,265,779;4,265,779; 3,455,839; 3,933,672; 4,652,392; 4,978,471; 4,983,316;5,288,431; 4,639,489; 4,749,740; and 4,798,679; 4,075,118; EuropeanPatent Application No. 89307851.9; EP 150,872; and DOS 2,124,526.

For any detergent compositions to be used in automatic laundry washingmachines, suds should not form to the extent that they overflow thewashing machine. Suds suppressors, when utilized, are preferably presentin a “suds suppressing amount. By “suds suppressing amount” is meantthat the formulator of the composition can select an amount of this sudscontrolling agent that will sufficiently control the suds to result in alow-sudsing laundry detergent for use in automatic laundry washingmachines. The compositions herein will generally comprise from 0% to 10%of suds suppressor. When utilized as suds suppressors, monocarboxylicfatty acids, and salts therein, will be present typically in amounts upto 5%, by weight, of the detergent composition. Preferably, from 0.5% to3% of fatty monocarboxylate suds suppressor is utilized. Silicone sudssuppressors are typically utilized in amounts up to 2.0%, by weight, ofthe detergent composition, although higher amounts may be used.Monostearyl phosphate suds suppressors are generally utilized in amountsranging from 0.1% to 2%, by weight, of the composition. Hydrocarbon sudssuppressors are typically utilized in amounts ranging from 0.01% to5.0%, although higher levels can be used. The alcohol suds suppressorsare typically used at 0.2%-3% by weight of the finished compositions.

Pearlescent Agents.

Pearlescent agents as described in WO2011/163457 may be incorporatedinto the compositions of the invention.

Perfume.

Preferably the composition comprises a perfume, preferably in the rangefrom 0.001 to 3 wt %, most preferably from 0.1 to 1 wt %. Many suitableexamples of perfumes are provided in the CTFA (Cosmetic, Toiletry andFragrance Association) 1992 International Buyers Guide, published byCFTA Publications and OPD 1993 Chemicals Buyers Directory 80^(th) AnnualEdition, published by Schnell Publishing Co. It is usual for a pluralityof perfume components to be present in the compositions of theinvention, for example four, five, six, seven or more. In perfumemixtures preferably 15 to 25 wt % are top notes. Top notes are definedby Poucher (Journal of the Society of Cosmetic Chemists 6(2):80 [1995]).Preferred top notes include rose oxide, citrus oils, linalyl acetate,lavender, linalool, dihydromyrcenol and cis-3-hexanol.

Processes of Making and Using Cleaning Compositions

The present invention also provides a method of making a composition asdescribed above comprising obtaining at least one of the variantsdescribed above and mixing it with an adjunct material or mixturethereof.

The cleaning compositions of the present invention are formulated intoany suitable form such as a solid, powder, liquid, tablet or gel and themixing of the variant and the adjunct may be by any suitable processchosen by the formulator, (See e.g., U.S. Pat. Nos. 5,879,584,5,691,297, 5,574,005, 5,569,645, 5,565,422, 5,516,448, 5,489,392,5,486,303, 4,515,705, 4,537,706, 4,515,707, 4,550,862, 4,561,998,4,597,898, 4,968,451, 5,565,145, 5,929,022, 6,294,514 and 6,376,445).

In some embodiments, the cleaning compositions of the present inventionare provided in unit dose form, including tablets, capsules, sachets,pouches, and multi-compartment pouches. In some embodiments, the unitdose format is designed to provide controlled release of the ingredientswithin a multi-compartment pouch (or other unit dose format). Suitableunit dose and controlled release formats are known in the art (See e.g.,EP 2 100 949, WO 02/102955, U.S. Pat. Nos. 4,765,916 and 4,972,017, andWO 04/111178 for materials suitable for use in unit dose and controlledrelease formats). In some embodiments, the unit dose form is provided bytablets wrapped with a water-soluble film or water-soluble pouches.Various formats for unit doses are provided in EP 2 100 947, and areknown in the art. In a preferred multi-compartment pouch, the proteasevariant is in a separate compartment from one or more of a furtherprotease enzyme, a first wash lipase enzyme, a cellulase enzyme, anamylase enzyme, a bleach component, a pH modifier, a fabric hueing dye,an optical brightener and a non-ionic surfactant, or mixtures thereof.

Methods of Use

The present invention also provides a method of treating a surface,particularly a fabric, comprising contacting the surface with an aqueousliquor comprising at least one of the variants listed above and anadjunct material Typically the protease variant and adjunct material areadded to water to form the wash liquor by adding the composition of theinvention to water. The surface to be treated in the method of theinvention may be any fabric or home care surface for cleaning such asdishware, laundry, hard surfaces, contact lenses, etc. In someembodiments, at least a portion of the surface is contacted with atleast one embodiment of the cleaning compositions of the presentinvention, in neat form or diluted in a wash liquor, and then thesurface is optionally washed and/or rinsed. For purposes of the presentinvention, “washing” includes, but is not limited to, soaking,scrubbing, and mechanical washing. In some embodiments, the cleaningcompositions of the present invention are used at concentrations of fromabout 500 ppm to about 15,000 ppm in solution. In some embodiments inwhich the wash solvent is water, the water temperature typically rangesfrom about 5° C. to about 90° C.

The present invention provides methods for cleaning or washing an itemor surface (e.g., hard surface) in need of cleaning, including, but notlimited to methods for cleaning or washing a dishware item, a tablewareitem, a fabric item, a laundry item, personal care item, etc., or thelike, and methods for cleaning or washing a hard or soft surface (e.g.,a hard surface of an item).

In some embodiments, the present invention provides a method forcleaning an item, object, or surface in need of cleaning, the methodcomprising contacting the item or surface (or a portion of the item orsurface desired to be cleaned) with at least one variant subtilisinprotease of the present invention or a composition of the presentinvention for a sufficient time and/or under conditions suitable and/oreffective to clean the item, object, or surface to a desired degree.Some such methods further comprise rinsing the item, object, or surfacewith water. For some such methods, the cleaning composition is adishwashing detergent composition and the item or object to be cleanedis a dishware item or tableware item. As used herein, a “dishware item”is an item generally used in serving or eating food. A dishware item canbe, but is not limited to for example, a dish, plate, cup, bowl, etc.,and the like. As used herein, “tableware” is a broader term thatincludes, but is not limited to for example, dishes, cutlery, knives,forks, spoons, chopsticks, glassware, pitchers, sauce boats, drinkingvessels, serving items, etc. It is intended that “tableware item”includes any of these or similar items for serving or eating food. Forsome such methods, the cleaning composition is an automatic dishwashingdetergent composition or a hand dishwashing detergent composition andthe item or object to be cleaned is a dishware or tableware item. Forsome such methods, the cleaning composition is a laundry detergentcomposition (e.g., a power laundry detergent composition or a liquidlaundry detergent composition), and the item to be cleaned is a fabricitem. In some other embodiments, the cleaning composition is a laundrypre-treatment composition. The cleaning composition may comprise a hardsurface cleaner.

In some embodiments, the present invention provides methods for cleaningor washing a fabric item optionally in need of cleaning or washing,respectively. In some embodiments, the methods comprise providing acomposition comprising the variant protease, including but not limitedto fabric or laundry cleaning composition, and a fabric item or laundryitem in need of cleaning, and contacting the fabric item or laundry item(or a portion of the item desired to be cleaned) with the compositionunder conditions sufficient or effective to clean or wash the fabric orlaundry item to a desired degree.

In some embodiments, the present invention provides a method forcleaning or washing an item or surface (e.g., hard surface) optionallyin need of cleaning, the method comprising providing an item or surfaceto be cleaned or washed and contacting the item or surface (or a portionof the item or surface desired to be cleaned or washed) with at leastone subtilisin variant of the invention or a composition of theinvention comprising at least one such subtilisin variant for asufficient time and/or under conditions sufficient or effective to cleanor wash the item or surface to a desired degree. Such compositionsinclude, but are not limited to for example, a cleaning composition ordetergent composition of the invention (e.g., a hand dishwashingdetergent composition, hand dishwashing cleaning composition, laundrydetergent or fabric detergent or laundry or fabric cleaning composition,liquid laundry detergent, liquid laundry cleaning composition, powderlaundry detergent composition, powder laundry cleaning composition,automatic dishwashing detergent composition, laundry booster cleaning ordetergent composition, laundry cleaning additive, and laundrypre-spotter composition, etc.). In some embodiments, the method isrepeated one or more times, particularly if additional cleaning orwashing is desired. For example, in some instance, the method optionallyfurther comprises allowing the item or surface to remain in contact withthe at least one variant protease or composition for a period of timesufficient or effective to clean or wash the item or surface to thedesired degree. In some embodiments, the methods further compriserinsing the item or surface with water and/or another liquid. In someembodiments, the methods further comprise contacting the item or surfacewith at least one variant protease of the invention or a composition ofthe invention again and allowing the item or surface to remain incontact with the at least one variant protease or composition for aperiod of time sufficient to clean or wash the item or surface to thedesired degree. In some embodiments, the cleaning composition is adishwashing detergent composition and the item to be cleaned is adishware or tableware item. In some embodiments of the present methods,the cleaning composition is an automatic dishwashing detergentcomposition or a hand dishwashing detergent composition and the item tobe cleaned is a dishware or tableware item. In some embodiments of themethods, the cleaning composition is a laundry detergent composition andthe item to be cleaned is a fabric item.

The present invention also provides methods of cleaning a tableware ordishware item in an automatic dishwashing machine, the method comprisingproviding an automatic dishwashing machine, placing an amount of anautomatic dishwashing composition comprising at least one subtilisinvariant of the present invention or a composition of the inventionsufficient to clean the tableware or dishware item in the machine (e.g.,by placing the composition in an appropriate or provided detergentcompartment or dispenser in the machine), putting a dishware ortableware item in the machine, and operating the machine so as to cleanthe tableware or dishware item (e.g., as per the manufacturer'sinstructions). In some embodiments, the methods include any automaticdishwashing composition described herein, which comprises, but is notlimited to at least one subtilisin variant provided herein. The amountof automatic dishwashing composition to be used can be readilydetermined according to the manufacturer's instructions or suggestionsand any form of automatic dishwashing composition comprising at leastone variant protease of the invention (e.g., liquid, powder, solid, gel,tablet, etc.), including any described herein, may be employed.

The present invention also provides methods for cleaning a surface, itemor object optionally in need of cleaning, the method comprisescontacting the item or surface (or a portion of the item or surfacedesired to be cleaned) with at least one variant subtilisin of thepresent invention or a cleaning composition of the invention in neatform or diluted in a wash liquor for a sufficient time and/or underconditions sufficient or effective to clean or wash the item or surfaceto a desired degree. The surface, item, or object may then be(optionally) washed and/or rinsed if desired. For purposes of thepresent invention, “washing” includes, but is not limited to forexample, scrubbing and mechanical agitation. In some embodiments, thecleaning compositions are employed at concentrations of from about 500ppm to about 15,000 ppm in solution (e.g., aqueous solution). When thewash solvent is water, the water temperature typically ranges from about5° C. to about 90° C. and when the surface, item or object comprises afabric, the water to fabric mass ratio is typically from about 1:1 toabout 30:1.

The present invention also provides methods of cleaning a laundry orfabric item in an washing machine, the method comprising providing anwashing machine, placing an amount of a laundry detergent compositioncomprising at least one variant subtilisin of the invention sufficientto clean the laundry or fabric item in the machine (e.g., by placing thecomposition in an appropriate or provided detergent compartment ordispenser in the machine), placing the laundry or fabric item in themachine, and operating the machine so as to clean the laundry or fabricitem (e.g., as per the manufacturer's instructions). The methods of thepresent invention include any laundry washing detergent compositiondescribed herein, comprising but not limited to at least one of anyvariant subtilisin provided herein. The amount of laundry detergentcomposition to be used can be readily determined according tomanufacturer's instructions or suggestions and any form of laundrydetergent composition comprising at least one variant protease of theinvention (e.g., solid, powder, liquid, tablet, gel, etc.), includingany described herein, may be employed.

EXPERIMENTAL

The present invention is described in further detail in the followingexamples which are not in any way intended to limit the scope of theinvention as claimed.

In the experimental disclosure which follows, the followingabbreviations apply: PI (Performance Index), ppm (parts per million); M(molar); mM (millimolar); μM (micromolar); nM (nanomolar); mol (moles);mmol (millimoles); pmol (micromoles); nmol (nanomoles); gm (grams); mg(milligrams); μg (micrograms); pg (picograms); L (liters); ml and mL(milliliters); μl and μL (microliters); cm (centimeters); mm(millimeters); μm (micrometers); nm (nanometers); U (units); V (volts);MW (molecular weight); sec (seconds); min(s) (minute/minutes); h(s) andhr(s) (hour/hours); ° C. (degrees Centigrade); QS (quantity sufficient);ND (not done); rpm (revolutions per minute); GH (degrees Germanhardness); H₂O (water); dH₂O (deionized water); HCl (hydrochloric acid);aa (amino acid); by (base pair); kb (kilobase pair); kD (kilodaltons);cDNA (copy or complementary DNA); DNA (deoxyribonucleic acid); ssDNA(single stranded DNA); dsDNA (double stranded DNA); dNTP(deoxyribonucleotide triphosphate); RNA (ribonucleic acid); MgCl₂(magnesium chloride); NaCl (sodium chloride); w/v (weight to volume);v/v (volume to volume); w/w (weight to weight); g (gravity); OD (opticaldensity); ppm (parts per million); Dulbecco's phosphate bufferedsolution (DPBS); SOC (2% Bacto-Tryptone, 0.5% Bacto Yeast Extract, 10 mMNaCl, 2.5 mM KCl); Terrific Broth (TB; 12 g/l Bacto-Tryptone, 24 g/lglycerol, 2.31 g/l KH₂PO₄, and 12.54 g/l K2HPO₄); OD₂₈₀ (optical densityat 280 nm); OD₆₀₀ (optical density at 600 nm); A₄₀₅ (absorbance at 405nm); Vmax (the maximum initial velocity of an enzyme catalyzedreaction); PAGE (polyacrylamide gel electrophoresis); PBS (phosphatebuffered saline [150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2]);PBST (PBS+0.25% TWEEN®-20); PEG (polyethylene glycol); PCR (polymerasechain reaction); RT-PCR (reverse transcription PCR); SDS (sodium dodecylsulfate); Tris (tris(hydroxymethyl)aminomethane); HEPES(N-[2-Hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]); HBS (HEPESbuffered saline); Tris-HCl(tris[Hydroxymethyl]aminomethane-hydrochloride); Tricine(N-[tris-(hydroxymethyl)-methyl]-glycine); CHES (2-(N-cyclo-hexylamino)ethane-sulfonic acid); TAPS(3-{[tris-(hydroxymethyl)-methyl]-amino}-propanesulfonic acid); CAPS(3-(cyclo-hexylamino)-propane-sulfonic acid; DMSO (dimethyl sulfoxide);DTT (1,4-dithio-DL-threitol); SA (sinapinic acid(s,5-dimethoxy-4-hydroxy cinnamic acid); TCA (trichloroacetic acid);Glut and GSH (reduced glutathione); GSSG (oxidized glutathione); TCEP(Tris[2-carboxyethyl]phosphine); Ci (Curies); mCi (milliCuries); μCi(microCuries); HPLC (high pressure liquid chromatography); RP-HPLC(reverse phase high pressure liquid chromatography); TLC (thin layerchromatography); MALDI-TOF (matrix-assisted laserdesorption/ionization—time of flight); Ts (tosyl); Bn (benzyl); Ph(phenyl); Ms (mesyl); Et (ethyl), Me (methyl); Tay (Thermus aquaticusDNA polymerase); Klenow (DNA polymerase I large (Klenow) fragment); EGTA(ethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid);EDTA (ethylenediaminetetracetic acid); bla (β-lactamase orampicillin-resistance gene); MJ Research (MJ Research, Reno, Nev.);Baseclear (Baseclear BV, Inc., Leiden, the Netherlands); PerSeptive(PerSeptive Biosystems, Framingham, Mass.); ThermoFinnigan(ThermoFinnigan, San Jose, Calif.); Argo (Argo BioAnalytica, MorrisPlains, N.J.); Seitz EKS (SeitzSchenk Filtersystems GmbH, Bad Kreuznach,Germany); Pall (Pall Corp., East Hills, N.Y. and Bad Kreuznach,Germany); Spectrum (Spectrum Laboratories, Dominguez Rancho, Calif.);Molecular Structure (Molecular Structure Corp., Woodlands, Tex.);Accelrys (Accelrys, Inc., San Diego, Calif.); Chemical Computing(Chemical Computing Corp., Montreal, Canada); New Brunswick (NewBrunswick Scientific, Co., Edison, N.J.); CFT (Center for TestMaterials, Vlaardingen, the Netherlands); P&G and Procter & Gamble(Procter & Gamble, Inc., Cincinnati, Ohio); GE Healthcare (GEHealthcare, Chalfont St. Giles, United Kingdom); DNA2.0 (DNA2.0, MenloPark, Calif.); OXOID (Oxoid, Basingstoke, Hampshire, UK); Megazyme(Megazyme International Ireland Ltd., Bray Business Park, Bray, Co.,Wicklow, Ireland); Finnzymes (Finnzymes Oy, Espoo, Finland); Kelco (CPKelco, Wilmington, Del.); Corning (Corning Life Sciences, Corning,N.Y.); (NEN (NEN Life Science Products, Boston, Mass.); Pharma AS(Pharma AS, Oslo, Norway); Dynal (Dynal, Oslo, Norway); Bio-Synthesis(Bio-Synthesis, Lewisville, Tex.); ATCC (American Type CultureCollection, Rockville, Md.); Gibco/BRL (Gibco/BRL, Grand Island, N.Y.);Sigma (Sigma Chemical Co., St. Louis, Mo.); Pharmacia (PharmaciaBiotech, Piscataway, N.J.); NCBI (National Center for BiotechnologyInformation); Applied Biosystems (Applied Biosystems, Foster City,Calif.); BD Biosciences and/or Clontech (BD Biosciences CLONTECHLaboratories, Palo Alto, Calif.); Operon Technologies (OperonTechnologies, Inc., Alameda, Calif.); MWG Biotech (MWG Biotech, HighPoint, N.C.); Oligos Etc (Oligos Etc. Inc, Wilsonville, Oreg.); Bachem(Bachem Bioscience, Inc., King of Prussia, Pa.); Difco (DifcoLaboratories, Detroit, Mich.); Mediatech (Mediatech, Herndon, Va.; SantaCruz (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.); Oxoid (OxoidInc., Ogdensburg, N.Y.); Worthington (Worthington Biochemical Corp.,Freehold, N.J.); GIBCO BRL or Gibco BRL (Life Technologies, Inc.,Gaithersburg, Md.); Millipore (Millipore, Billerica, Mass.); Bio-Rad(Bio-Rad, Hercules, Calif.); Invitrogen (Invitrogen Corp., San Diego,Calif.); NEB (New England Biolabs, Beverly, Mass.); Sigma (SigmaChemical Co., St. Louis, Mo.); Pierce (Pierce Biotechnology, Rockford,Ill.); Takara (Takara Bio Inc. Otsu, Japan); Roche (Hoffmann-La Roche,Basel, Switzerland); EM Science (EM Science, Gibbstown, N.J.); Qiagen(Qiagen, Inc., Valencia, Calif.); Biodesign (Biodesign Intl., Saco,Me.); Aptagen (Aptagen, Inc., Herndon, Va.); Sorvall (Sorvall brand,from Kendro Laboratory Products, Asheville, N.C.); Molecular Devices(Molecular Devices, Corp., Sunnyvale, Calif.); R&D Systems (R&D Systems,Minneapolis, Minn.); Siegfried Handel (Siegfried Handel AG, Zofingen,Switzerland); Stratagene (Stratagene Cloning Systems, La Jolla, Calif.);Marsh (Marsh Biosciences, Rochester, N.Y.); Geneart (Geneart GmbH,Regensburg, Germany); Bio-Tek (Bio-Tek Instruments, Winooski, Vt.);(Biacore (Biacore, Inc., Piscataway, N.J.); PeproTech (PeproTech, RockyHill, N.J.); SynPep (SynPep, Dublin, Calif.); New Objective (NewObjective brand; Scientific Instrument Services, Inc., Ringoes, N.J.);Waters (Waters, Inc., Milford, Mass.); Matrix Science (Matrix Science,Boston, Mass.); Dionex (Dionex, Corp., Sunnyvale, Calif.); Monsanto(Monsanto Co., St. Louis, Mo.); Wintershall (Wintershall AG, Kassel,Germany); BASF (BASF Co., Florham Park, N.J.); Huntsman (HuntsmanPetrochemical Corp., Salt Lake City, Utah); Shell Chemicals (ShellChemicals, Inc., London, UK); Stepan (Stepan, Northfield, Ill.);Clariant (Clariant, Sulzbach, Germany); Industrial Zeolite (IndustrialZeolite Ltd., Grays, Essex, UK); Jungbunzlauer (Jungbunzlauer, Basel,Switzerland); Solvay (Solvay, Brussels, Belgium); 3V Sigma (3V Sigma,Bergamo, Italy); Innospec (Innospec, Ellesmere Port, UK); Thermphos(Thermphos, Vlissiggen-Ost, the Netherlands); Ciba Specialty (CibaSpecialty Chemicals, Basel, Switzerland); Dow Corning (Dow Corning,Barry, UK); Enichem (Enichem Iberica, Barcelona, Spain); Fluka Chemie AG(Fluka Chemie AG, Buchs, Switzerland); Gist-Brocades (Gist-Brocades, NV,Delft, the Netherlands); Dow Corning (Dow Corning Corp., Midland,Mich.); Mettler-Toledo (Mettler-Toledo Inc, Columbus, Ohio); RB(Reckitt-Benckiser, Slough, UK); and Microsoft (Microsoft, Inc.,Redmond, Wash.).

As used herein, in some lists, a leading “0” is indicated, in order toprovide a three number designation for each site (e.g., “001” is thesame as “1,” so “A001C” is the same as “A1C”). In some lists, theleading “0” is not included. In addition, as used herein, “X” refers toany amino acid.

In the exemplified detergent compositions provided herein, the enzymeslevels are expressed by pure enzyme by weight of the total compositionand unless otherwise specified, the detergent ingredients are expressedby weight of the total compositions. The abbreviated componentidentifications therein have the following meanings:

Abbreviation Ingredient LAS Sodium linear C₁₁₋₁₃ alkyl benzenesulfonate. NaC16-17HSAS Sodium C₁₆₋₁₇ highly soluble alkyl sulfate TASSodium tallow alkyl sulphate. CxyAS Sodium C_(1x)-C_(1y) alkyl sulfate.CxyEz C_(1x)-C_(1y) predominantly linear primary alcohol condensed withan average of z moles of ethylene oxide. CxyAEzS C_(1x)-C_(1y) sodiumalkyl sulfate condensed with an average of z moles of ethylene oxide.Added molecule name in the examples. Nonionic Mixedethoxylated/propoxylated fatty alcohol e.g. Plurafac LF404 being analcohol with an average degree of ethoxylation of 3.8 and an averagedegree of propoxylation of 4.5. QAS R₂•N + (CH₃)₂(C₂H₄OH) with R₂ =C₁₂-C₁₄. Silicate Amorphous Sodium Silicate (SiO₂:Na₂O ratio =1.6-3.2:1). Metasilicate Sodium metasilicate (SiO₂:Na₂O ratio = 1.0).Zeolite A Hydrated aluminosilicate of formula Na₁₂(AlO₂SiO₂)₁₂•27H₂OSKS-6 Crystalline layered silicate of formula δ-Na₂Si₂O₅. SulfateAnhydrous sodium sulphate. STPP Sodium Tripolyphosphate. MA/AA Randomcopolymer of 4:1 acrylate/maleate, average molecular weight about70,000-80,000. AA Sodium polyacrylate polymer of average molecularweight 4,500. Polycarboxylate Copolymer comprising mixture ofcarboxylated monomers such as acrylate, maleate and methyacrylate with aMW ranging between 2,000-80,000 such as Sokolan commercially availablefrom BASF, being a copolymer of acrylic acid, MW4,500. BB13-(3,4-Dihydroisoquinolinium)propane sulfonate BB21-(3,4-dihydroisoquinolinium)-decane-2-sulfate PB1 Sodium perboratemonohydrate. PB4 Sodium perborate tetrahydrate of nominal formulaNaBO₃•4H₂O. Percarbonate Sodium percarbonate of nominal formula2Na₂CO₃•3H₂O₂. TAED Tetraacetyl ethylene diamine. NOBSNonanoyloxybenzene sulfonate in the form of the sodium salt. DTPADiethylene triamine pentaacetic acid. HEDP 1,1-hydroxyethanediphosphonic acid. DETPMP Diethyltriamine penta (methylene) phosphonate,marketed by Monsanto under the Trade name Dequest 2060. EDDSEthylenediamine-N,N′-disuccinic acid, (S,S) isomer in the form of itssodium salt Diamine Dimethyl aminopropyl amine; 1,6-hezane diamine;1,3-propane diamine; 2-methyl-1,5-pentane diamine; 1,3-pentanediamine;1- methyl-diaminopropane. DETBCHD 5,12-diethyl-1,5,8,12-tetraazabicyclo[6,6,2] hexadecane, dichloride, Mn(II) SALT PAAC Pentaamine acetatecobalt(III) salt. Paraffin Paraffin oil sold under the tradename Winog70 by Wintershall. Paraffin Sulfonate A Paraffin oil or wax in whichsome of the hydrogen atoms have been replaced by sulfonate groups.Aldose oxidase Oxidase enzyme sold under the tradename Aldose Oxidase byNovozymes A/S Galactose oxidase Galactose oxidase from Sigma nprE Therecombinant form of neutral metalloprotease expressed in Bacillussubtilis (See e.g., WO 07/044993) PMN Purified neutral metalloproteasefrom Bacillus amyloliquefacients. Amylase A suitable amylolytic enzyme,such as those sold under the tradenames PURAFECT ® Ox described in WO94/18314, WO96/05295 sold by Genencor; NATALASE ®, TERMAMYL ®,FUNGAMYI ® and DURAMYL ™, all available from Novozymes A/S. Lipase Asuitable lipolytic enzyme such as those sold under the tradenamesLIPEX ®, LIPOLASE ®, LIPOLASE ® Ultra by Novozymes A/S and Lipomax ™ byGist-Brocades. Cellulase A suitable cellulytic enzyme such as those soldunder the tradenames CAREZYME ®, CELLUZYME ®, and/or ENDOLASE ® byNovozymes A/S. Pectin Lyase A suitable pectin lyase, such as those soldunder the tradenames PECTAWAY ® and PECTAWASH ® available from NovozymesA/S. PVP Polyvinylpyrrolidone with an average molecular weight of 60,000PVNO Polyvinylpyridine-N-Oxide, with an average molecular weight of50,000. PVPVI Copolymer of vinylimidazole and vinylpyrrolidone, with anaverage molecular weight of 20,000. Brightener 1 Disodium4,4′-bis(2-sulphostyryl)biphenyl. Silicone antifoam Polydimethylsiloxanefoam controller with siloxane-oxyalkylene copolymer as dispersing agentwith a ratio of said foam controller to said dispersing agent of 10:1 to100:1. Suds Suppressor 12% Silicone/silica, 18% stearyl alcohol, 70%starch in granular form. SRP 1 Anionically end capped poly esters. PEG XPolyethylene glycol, of a molecular weight of x. PVP K60 ®Vinylpyrrolidone homopolymer (average MW 160,000) Jeffamine ® ED-2001Capped polyethylene glycol from Huntsman Isachem ® AS A branched alcoholalkyl sulphate from Enichem MME PEG (2000) Monomethyl ether polyethyleneglycol (MW 2000) from Fluka Chemie AG. DC3225C Silicone suds suppresser,mixture of Silicone oil and Silica from Dow Corning. TEPAETetreaethylenepentaamine ethoxylate. BTA Benzotriazole. Betaine(CH₃)₃N⁺CH₂COO⁻ Sugar Industry grade D-glucose or food grade sugar CFAAC₁₂-C₁₄ alkyl N-methyl glucamide TPKFA C₁₂-C₁₄ topped whole cut fattyacids. Clay A hydrated aluminumu silicate in a general formulaAl₂O₃SiO₂•xH₂O. Types: Kaolinite, montmorillonite, atapulgite, illite,bentonite, halloysite. pH Measured as a 1% solution in distilled waterat 20° C.

For North American (NA) and Western European (WE) heavy duty liquidlaundry (HDL) detergents, heat inactivation of the enzymes present incommercially-available detergents is performed by placing pre-weighedliquid detergent (in a glass bottle) in a water bath at 95° C. for 2hours. The incubation time for heat inactivation of NA and WE auto dishwashing (ADW) detergents is 8 hours. Both un-heated and heateddetergents are assayed within 5 minutes of dissolving the detergent toaccurately determine percentage deactivated. Enzyme activity is testedby the AAPF assay.

For testing of enzyme activity in heat-inactivated detergents, workingsolutions of detergents are made from the heat inactivated stocks.Appropriate amounts of water hardness (e.g., 6 gpg or 12 gpg) and bufferare added to the detergent solutions to match the desired conditions.The solutions are mixed by vortexing or inverting the bottles. Thefollowing Table A provides information regarding some detergentcompositions. In some experiments, additional and/or other commerciallyavailable detergents find use in the following Examples.

Table A provides granular laundry detergent compositions produced inaccordance with the invention suitable for laundering fabrics.

TABLE A Granular Laundry Detergent Compositions and Their ComponentsDetergent Compositions Component 1 2 3 4 5 6 Linearalkylbenzenesulfonate 15 12 20 10 12 13 with aliphatic carbon chainlength C₁₁—C₁₂ Other surfactants 1.6 1.2 1.9 3.2 0.5 1.2 Phosphatebuilder(s) 2 3 4 Zeolite 1 1 4 1 Silicate 4 5 2 3 3 5 Sodium Carbonate 25 5 4 0 3 Polyacrylate (MW 4500) 1 0.6 1 1 1.5 1 Carboxymethyl cellulose1 — 0.3 — 1.1 — (Finnfix BDA ex CPKelco) Celluclean ® (15.6 mg/g) 0.230.17 0.5 0.2 0.2 0.6 Cold Water Protease* 0.23 0.17 0.05 0.2 0.03 0.1Stainzyme Plus ® (14 mg/g) 0.23 0.17 0.5 0.2 0.2 0.6 Mannaway 4.0T (4mg/g) 0.1 0.1 0.1 Lipex 100T (18.6 mg/g) 0.2 0.1 0.3 FluorescentBrightener(s) 0.16 0.06 0.16 0.18 0.16 0.16 Diethylenetriamine 0.6 0.60.25 0.6 0.6 pentaacetic acid or Ethylene diamine tetraacetic acid MgSO₄1 1 1 0.5 1 1 Bleach(es) and Bleach 6.88 6.12 2.09 1.17 4.66activator(s) Ethoxylated thiophene Hueing 0.002 0.001 0.003 0.003 — —Dye⁵ Direct Violet 9 ex Ciba 0.0006 0.0004 0.0006 Specialty ChemicalsSulfate/Citric Acid/Sodium Balance to 100% Bicarbonate/ Moisture/perfume¹Random graft copolymer is a polyvinyl acetate grafted polyethyleneoxide copolymer having a polyethylene oxide backbone and multiplepolyvinyl acetate side chains. The molecular weight of the polyethyleneoxide backbone is about 6000 and the weight ratio of the polyethyleneoxide to polyvinyl acetate is about 40 to 60 and no more than 1 graftingpoint per 50 ethylene oxide units. ²Polyethylenimine (MW = 600) with 20ethoxylate groups per —NH. ³Amphiphilic alkoxylated grease cleaningpolymer is a polyethylenimine (MW = 600) with 24 ethoxylate groups per—NH and 16 propoxylate groups per —NH ⁴Reversible protease inhibitor ofstructure:

⁵Ethoxylated thiophene Hueing Dye is as described in U.S. Pat. No.7,208,459 B2.

In Table A, all enzyme levels expressed as % enzyme raw material, exceptfor cold water protease (of this invention) which is expressed as % ofactive protein added to the product.

Table B provides granular laundry detergent compositions suitable fortop-loading automatic washing machines (detergent compositions 7-9) andfront loading washing machines (detergent compositions 10-11). The GG36protease variant tested and/or cold water protease of the presentinvention is added separately to these formulations.

TABLE B Granular Laundry Detergent Compositions and Their ComponentsDetergent Composition Component 7 8 9 10 11 Surfactants C₁₆₋₁₇ Branchedalkyl sulfate 3.55 15.8 C₁₂₋₁₄ alkyl sulphate 1.5 Sodium linearalkylbenzenesulfonate 9.6 10.6 7.5 9 with aliphatic chain length C₁₁-C₁₂Sodium C_(14/15) alcohol ethoxy-3- 1.15 2.88 sulfate Sodium C_(14/15)alkyl sulphate 2.37 C_(14/15) alcohol ethoxylate with average 7 1.17 1moles of ethoxylation mono-C₈₋₁₀ alkyl mono-hydroxyethyl di- 0.45 methylquaternary ammonium chloride Di methyl hydroxyl ethyl lauryl 0.18ammonium chloride Zeolite A 13.9 4.7 0.01 2.9 1.8 Sodium Silicate1.6.ratio 4 0.2 4 4 Sodium Silicate 2.35.ratio 8 Citric Acid 2.5 1.4Sodium tripolyphosphate 5 Sodium Carbonate 24.1 30 16.9 24.4 21Nonanoyloxybenzenesuplhonate 5.78 2.81 0.96 Oxaziridinium-based bleachbooster 0.03 0.017 Tetrasodium S,S,- 0.2 ethylenediaminedisuccinateDiethylenetriamine penta (methylene 0.61 0.33 phosphonic acid),heptasodium salt Hydroxyethane dimethylene phosphonic 0.29 0.45 acidEthylene diamine tetraacetate 0.27 MgSO4 0.47 0.5994 0.782 SodiumPercarbonate 7 4.4 15.9 19.1 Tetra Acetyl Ethylene Diamine 3.3 4.6Sodium Perborate Monohydrate 1.2 Carboxymethyl cellulose 0.1 0.17 1.690.23 (e.g. Finnfix BDA ex CPKelco) Sodium Acrylic acid/maleic acid co-0.0236 3.8 2 2.5 polymer (70/30) Sodium polyacrylate (Sokalan PA30 CL) 40.84 Terephthalate polymer 0.23 Polyethylene glycol/vinyl acetate 0.890.89 0.91 random graft co polymer Photobleach-zinc phthalocyanine 0.0050.001 0.002 tetrasulfonate C.I.Fluorescent Brightener 260 0.11 0.15 0.040.23 0.15 C.I.Fluorescent Brightener 351 (Tinopal ® 0.1 CBS) Sudssuppressor granule 0.25 0.07 0.04 Hydrophobically modified carboxy 0.0190.028 methyl cellulose (Finnifix ® SH-1) Bentonite 8.35 Miscellaneous(Dyes, perfumes, process Balance Balance Balance Balance Balance aids,moisture and sodium sulphate)

In Table B, surfactant ingredients can be obtained from any suitablesupplier, including but not limited to BASF (e.g., LUTENSOL®), ShellChemicals, Stepan, Huntsman, and Clariant (e.g., PRAEPAGEN®). Zeolitecan be obtained from sources such as Industrial Zeolite. Citric acid andsodium citrate can be obtained from sources such as Jungbunzlauer.Sodium percarbonate, sodium carbonate, sodium bicarbonate and sodiumsesquicarbonate can be obtained from sources such as Solvay.Acrylate/maleate copolymers can be obtained from sources such as BASF.Carboxymethylcellulose and hydrophobically modified carboxymethylcellulose can be obtained from sources such as CPKelco. C.I. FluorescentBrightener 260 can be obtained from 3V Sigma (e.g., OPTIBLANC®,OPTIBLANC® 2M/G, OPTIBLANC®0.2MG/LT Extra, or OPTIBLANC® Ecobright.Tetrasodium S,S-ethylenediamine disuccinate can be obtained from sourcessuch as Innospec. Terephthalate co-polymer can be obtained from Clariant(e.g., REPELOTEX SF 2). In addition, 1-Hydroxyethane-1,1-diphosphonicacid can be obtained from Thermphos. Oxaziridinium-based bleach boosterhas the following structure, where R1=2-butyloctyl, and was producedaccording to US 2006/0089284A1.

The enzymes NATALASE®, TERMAMYL®, STAINZYME PLUS®, CELLUCLEAN® andMANNAWAY®, can be obtained from Novozymes. Zinc phthalocyaninetetrasulfonate can be obtained from Ciba Specialty Chemicals (e.g.,TINOLUX® BMC). Suds suppressor granule can be obtained from Dow Corning.In these detergent compositions, random graft copolymer is a polyvinylacetate grafted polyethylene oxide copolymer having a polyethylene oxidebackbone and multiple polyvinyl acetate side chains. The molecularweight of the polyethylene oxide backbone is about 6000 and the weightratio of the polyethylene oxide to polyvinyl acetate is about 40 to 60and no more than 1 grafting point per 50 ethylene oxide units.

Tables C-E provide additional granular detergent compositions suitablefor washing machines (detergents 36a-n). The GG36 protease varianttested or cold water protease of the present invention is addedseparately to these formulations.

TABLE C Additional Granular Laundry Detergent Compositions and TheirComponents Deterent Composition Component 36a 36b 36c 36d 36eSurfactants C₁₀ Nonionic 0.1843 C₁₆₋₁₇ Branched alkyl sulfate 3.53 3.533.53 C₁₂₋₁₄ alkyl sulphate Sodium linear alkylbenzenesulfonate 8.98 8.988.98 13.58 14.75 with aliphatic chain length C₁₁-C₁₂ Sodium C_(14/15)alcohol ethoxy-3- 1.28 1.28 1.28 sulfate Sodium C_(14/15) alkyl sulphate2.36 2.36 2.36 C_(14/15) alcohol ethoxylate with average 7 moles ofethoxylation mono-C₈₋₁₀ alkyl mono-hydroxyethyl di- methyl quaternaryammonium chloride Di methyl hydroxyl ethyl lauryl 0.1803 ammoniumchloride Zeolite A 15.31 15.31 15.31 4.47 Bentonite 8.35 Sodium Silicate1.6.ratio 0.16 Sodium Silicate 2.0.ratio 3.72 3.72 3.72 8.41 SodiumSilicate 2.35.ratio Citric Acid 0.0066 Sodium tripolyphosphate 5.06Sodium Carbonate 26.1 26.18 26.1 15.9 29.0 Nonanoyloxybenzenesuplhonate5.78 5.78 5.78 1.17 1.86 Oxaziridinium-based bleach booster 0.037 0.0370.037 Tetrasodium S,S,- ethylenediaminedisuccinate Diethylenetriaminepenta (methylene 0.62 0.62 0.62 phosphonic acid), heptasodium saltHydroxyethane dimethylene phosphonic acid Ethylene diamine tetraacetate0.2701 MgSO4 0.056 0.056 0.056 0.47 Sodium Percarbonate 7.06 7.06 3.64Tetra Acetyl Ethylene Diamine Sodium Perborate Monohydrate 1.47Carboxymethyl cellulose 0.38 0.38 0.38 0.173 (e.g. Finnfix BDA exCPKelco) Sodium Acrylic acid/maleic acid co- 3.79 3.78 3.79 3.64 polymer(70/30) Sodium polyacrylate (Sokalan PA30 CL) 3.78 3.78 3.78 0.842Terephthalate polymer Polyethylene glycol/vinyl acetate 0.89 randomgraft co polymer Photobleach-zinc phthalocyanine tetrasulfonateC.I.Fluorescent Brightener 260 0.1125 0.1125 0.1125 0.043 0.15C.I.Fluorescent Brightener 351 (Tinopal ® 0.0952 CBS) Suds suppressorgranule 0.015 0.015 0.015 0.031 Hyrdophobically modified carboxy methylcellulose (Finnifix ® SH-1) Bentonite Miscellaneous (Dyes, perfumes,process Balance Balance Balance Balance Balance aids, moisture andsodium sulphate)

TABLE D Additional Granular Laundry Detergent Compositions and TheirComponents Detergent Composition Component 36f 36g 36h 36i 36jSurfactants C₁₀ Nonionic 0.1142 0.2894 0.1885 0.1846 0.1885 C₁₆₋₁₇Branched alkyl sulfate C₁₂₋₁₄ alkyl sulphate Sodium linearalkylbenzenesulfonate 12.94 15.69 9.01 8.42 9.51 with aliphatic chainlength C₁₁-C₁₂ Sodium C_(14/15) alcohol ethoxy-3- sulfate SodiumC_(14/15) alkyl sulphate C_(12/14) alcohol ethoxylate with average 7 2.9moles of ethoxylation C_(12/14) alcohol ethoxylate with average 3 2.44moles of ethoxylation C_(14/15) alcohol ethoxylate with average 7 0.971.17 0.97 moles of ethoxylation mono-C₈₋₁₀ alkyl mono-hydroxyethyl di-0.45 methyl quaternary ammonium chloride Di methyl hydroxyl ethyl lauryl0.195 0.45 ammonium chloride Zeolite A 2.01 0.39 1.83 2.58 0.59 SodiumSilicate 1.6.ratio 4.53 5.62 4.53 Sodium Silicate 2.0.ratio 10.1 SodiumSilicate 2.35.ratio 7.05 Citric Acid 1.4 1.84 1.0 Sodiumtripolyphosphate 5.73 Sodium Carbonate 12.65 15.93 21.0 27.31 20.2Nonanoyloxybenzenesuplhonate 1.73 Oxaziridinium-based bleach booster0.0168 0.0333 0.024 Tetrasodium S,S,- ethylenediaminedisuccinateDiethylenetriamine penta (methylene 0.327 0.3272 phosphonic acid),heptasodium salt Hydroxyethane dimethylene phosphonic 0.45 0.2911 0.45acid Ethylene diamine tetraacetate 0.28 0.1957 MgSO4 0.54 0.79 0.64940.793 Sodium Percarbonate 19.1 15.85 22.5 Tetra Acetyl Ethylene Diamine4.554 3.71 5.24 Sodium Perborate Monohydrate 5.55 Carboxymethylcellulose 0.62 0.21 0.23 1.07 0.2622 (e.g. Finnfix BDA ex CPKelco)Sodium Acrylic acid/maleic acid co- 0.40 2.61 2.5 2.00 1.75 polymer(70/30) Sodium polyacrylate (Sokalan PA30 CL) 0.0055 0.011 0.008Terephthalate polymer 0.231 Polyethylene glycol/vinyl acetate 0.55 1.400.911 0.8924 0.911 random graft co polymer Photobleach-zincphthalocyanine tetrasulfonate C.I.Fluorescent Brightener 260 0.11740.048 0.1455 0.2252 0.1455 C.I.Fluorescent Brightener 351 (Tinopal ®0.1049 CBS) Suds suppressor granule 0.04 0.0658 0.04 Hyrdophobicallymodified carboxy methyl cellulose (Finnifix ® SH-1) BentoniteMiscellaneous (Dyes, perfumes, process Balance Balance Balance BalanceBalance aids, moisture and sodium sulphate)

TABLE E Additional Granular Laundry Detergent Compositions and TheirComponents Detergent Composition Component 36k 36l 36m 36n SurfactantsC₁₀ Nonionic 0.1979 0.1979 0.1979 0.1979 C₁₆₋₁₇ Branched alkyl sulfateC₁₂₋₁₄ alkyl sulphate Sodium linear alkylbenzenesulfonate 8.92 8.92 11.511.5 with aliphatic chain length C₁₁-C₁₂ Sodium C_(14/15) alcoholethoxy-3- 1.62 1.62 1.125 1.125 sulfate Sodium C_(14/15) alkyl sulphateC_(14/15) alcohol ethoxylate with average 7 1.0 1.0 1.5 1.5 moles ofethoxylation mono-C₈₋₁₀ alkyl mono-hydroxyethyl di- methyl quaternaryammonium chloride Di methyl hydroxyl ethyl lauryl ammonium chlorideZeolite A 1.63 1.63 2.0 2.0 Sodium Silicate 1.6.ratio 4.75 4.75 4.754.75 Sodium Silicate 2.0.ratio 0.06 0.06 Sodium Silicate 2.35.ratioCitric Acid 1.10 1.10 1.1 1.1 Sodium tripolyphosphate Sodium Carbonate23.3 23.3 23.3 23.3 Nonanoyloxybenzenesuplhonate Oxaziridinium-basedbleach booster 0.021 0.021 0.015 0.015 Tetrasodium S,S,- 0.26 0.26 0.260.26 ethylenediaminedisuccinate Diethylenetriamine penta (methylenephosphonic acid), heptasodium salt Hydroxyethane dimethylene phosphonic0.47 0.47 0.47 0.47 acid Ethylene diamine tetraacetate MgSO4 0.83 0.830.82 0.82 Sodium Percarbonate 19.35 19.35 19.35 19.35 Tetra AcetylEthylene Diamine 4.51 4.51 4.51 4.51 Sodium Perborate MonohydrateCarboxymethyl cellulose 1.01 1.01 1.01 1.01 (e.g. Finnfix BDA exCPKelco) Sodium Acrylic acid/maleic acid co- 1.84 1.84 1.84 1.84 polymer(70/30) Sodium polyacrylate (Sokalan PA30 CL) 0.007 0.007 0.005 0.005Terephthalate polymer 0.179 0.179 0.179 0.179 Polyethylene glycol/vinylacetate 0.96 0.96 0.96 0.96 random graft co polymer Photobleach-zincphthalocyanine tetrasulfonate C.I.Fluorescent Brightener 260 0.153 0.1530.171 0.171 C.I.Fluorescent Brightener 351 (Tinopal ® CBS) Sudssuppressor granule 0.042 0.042 0.042 0.042 Hyrdophobically modifiedcarboxy methyl cellulose (Finnifix ® SH-1) Bentonite Miscellaneous(Dyes, perfumes, process Balance Balance Balance Balance Balance aids,moisture and sodium sulphate)Notes for detergent compositions 36 a-n in Tables C, D, E:Surfactant ingredients can be obtained from BASF, Ludwigshafen, Germany(Lutensol®); Shell Chemicals, London, UK; Stepan, Northfield, Ill., USA;Huntsman, Huntsman, Salt Lake City, Utah, USA; Clariant, Sulzbach,Germany (Praepagen®).Zeolite can be obtained from Industrial Zeolite (UK) Ltd, Grays, Essex,UK.Citric acid and sodium citrate can be obtained from Jungbunzlauer,Basel, Switzerland.Sodium percarbonate, sodium carbonate, sodium bicarbonate and sodiumsesquicarbonate can be obtained from Solvay, Brussels, Belgium.Acrylate/maleate copolymers can be obtained from BASF, Ludwigshafen,Germany.Carboxymethylcellulose and hydrophobically modified carboxymethylcellulose can be obtained from CPKelco, Arnhem, The Netherlands.C.I. Fluorescent Brightener 260 can be obtained from 3V Sigma, Bergamo,Italy as Optiblanc® Optiblanc® 2M/G, Optiblanc® 2MG/LT Extra, orOptiblanc® Ecobright.Tetrasodium S,S-ethylenediamine disuccinate can be obtained fromInnospec, Ellesmere Port, UK.Terephthalate co-polymer can be obtained from Clariant under thetradename Repelotex SF 2. 1-Hydroxyethane-1,1-diphosphonic acid can beobtained from Thermphos, Vlissingen-Oost, The Netherlands.Oxaziridinium-based bleach booster has the following structure, whereR1=2-butyloctyl, and was produced according to US 2006/0089284A1.

Enzymes Natalase®, Termamyl®, Stainzyme Plus®, Celluclean® andMannaway®, can be obtained from Novozymes, Bagsvaerd, Denmark.Zinc phthalocyanine tetrasulfonate can be obtained from Ciba SpecialtyChemicals, Basel, Switzerland, as Tinolux® BMC.Suds suppressor granule can be obtained from Dow Corning, Barry, UK.Random graft copolymer is a polyvinyl acetate grafted polyethylene oxidecopolymer having a polyethylene oxide backbone and multiple polyvinylacetate side chains. The molecular weight of the polyethylene oxidebackbone is about 6000 and the weight ratio of the polyethylene oxide topolyvinyl acetate is about 40 to 60 and no more than 1 grafting pointper 50 ethylene oxide units.

Example 1 Assays and Test Methods

This Example describes the various Test Methods and assays used in thedevelopment of the present invention. Any deviations from the protocolsprovided are indicated in the pertinent Examples.

The assays were performed using a Biomek FX Robot (Beckman Coulter) or amultichannel pipettor (e.g., Rainin PipetLite, Mettler-Toledo) and aSpectraMAX MTP Reader (type 340; Molecular Devices).

A. Test Methods

Test Method 1

A protocol to define whether a dye or pigment material is a fabrichueing agent for the purpose of the invention is provided below:

-   1) Fill two tergotometer pots with 800 ml of Newcastle upon Tyne,    UK, City Water (−12 grains per US gallon total hardness, supplied by    Northumbrian Water, Pity Me, Durham, Co. Durham, UK).-   2) Insert pots into tergotometer, with water temperature controlled    at 30° C. and agitation set at 40 rpm for the duration of the    experiment.-   3) Add 4.8 g of IEC-B detergent (IEC 60456 Washing Machine Reference    Base Detergent Type B), supplied by wfk, Briiggen-Bracht, Germany,    to each pot.-   4) After two minutes, add 2.0 mg active colorant to the first pot.-   5) After one minute, add 50 g of flat cotton vest (supplied by    Warwick Equest, Consett, County Durham, UK), cut into 5 cm×5 cm    swatches, to each pot.-   6) After 10 minutes, drain the pots and re-fill with cold Water (16°    C.) having a water hardness of 14.4 English Clark Degrees Hardness    with a 3:1 Calcium to Magnesium molar ratio.-   7) After 2 minutes rinsing, remove fabrics.-   8) Repeat steps 3-7 for a further three cycles using the same    treatments.-   9) Collect and line dry the fabrics indoors for 12 hours.-   10) Analyse the swatches using a Hunter Miniscan spectrometer fitted    with D65 illuminant and UVA cutting filter, to obtain Hunter a    (red-green axis) and Hunter b (yellow-blue axis) values.-   11) Average the Hunter a and Hunter b values for each set of    fabrics. If the fabrics treated with colorant under assessment show    an average difference in hue of greater than 0.2 units on either the    a axis or b axis, it is deemed to be a fabric hueing agent for the    purpose of the invention.    Test Method 2

For Test Method 2, the BMI microswatch assay provided below is run usingthe granular detergent composition 10 (See Table D, above). The laundrydetergent is dissolved in water that has a hardness of 12 gpg andadjusted to a temperature of 16° C., and the protease variant enzyme ofinterest is added. Performance of the protease variant enzymes is thendetermined as per the BMI microswatch assay described. The performanceindex is determined by comparing the performance of the protease variantenzyme with that of the B. lentus GG36 subtilisin enzyme having theamino acid sequence of SEQ ID NO:2, with in all cases the enzyme dosagerange being 0.1-5 ppm. Protease variant enzymes having a performanceindex of 1.1 or greater are viewed to be cold water proteases.

Test Method 3

For Test Method 3, the BMI microswatch assay provided below is run usingthe granular laundry detergent composition 7 (See Table D, above). Thelaundry detergent is dissolved in water that has a hardness of 6 gpg andadjusted to a temperature of 16° C., the GG36 protease variant enzyme ofinterest is added. Performance of the GG36 protease variant enzymes isthen determined as per the BMI microswatch assay described. Theperformance index is determined by comparing the performance of the GG36protease variant enzyme with that of the B. lentus GG36 subtilisinenzyme having the amino acid sequence of SEQ ID NO:2, with in all casesthe enzyme dosage range being 0.1-5 ppm. GG36 protease variant enzymeshaving a performance index of 1.1 or greater are viewed to be cold waterproteases.

Test Method 4

For Test Method 4, the BMI microswatch assay is run using the granularlaundry detergent composition 7 (See Table D, above). The laundrydetergent is dissolved in water that has a hardness of 6 gpg andadjusted to a temperature of 16° C., and the GG36 protease variantenzyme of interest is added. Performance of the GG36 protease variantenzymes is then determined as per the BMI microswatch assay described.The performance index is determined by comparing the performance of theGG36 protease variant enzyme with that of a reference enzyme GG36-A158E,said GG36-A158E reference enzyme consisting of the B. lentus subtilisinGG36 protease amino acid sequence of SEQ ID NO:2 with a singlesubstitution of glutamic acid for alanine at position 158 (i.e., theA158E mutation), with in all cases the enzyme dosage range being 0.1-5ppm. GG36 protease variant enzymes having a performance index of 1.0 orgreater are viewed to be cold water proteases.

Test Method 6

For Test Method 6, the BMI microswatch assay is run using one of thedetergents 36a 36n in Table 1-2. The detergent is dissolved in waterthat has a hardness as specified in Table 1-2 and adjusted to atemperature of 16° C. Performance of the variant enzymes is thendetermined as per the BMI microswatch assay described. The performanceindex is determined by comparing the performance of the variant withthat of the enzyme of SEQ ID NO:2, with the enzyme dosage range being0.1-5 ppm in all cases. Enzymes having a performance index of 1.1 orgreater are viewed to be cold water proteases.

Test Method 7

For Test Method 7, the BMI microswatch assay is run using one of thedetergents in Table 1. The detergent is dissolved in water that has ahardness and buffer as specified in Table 1 and adjusted to atemperature of 16° C. or 25° C. Performance of the variant enzymes isthen determined as per the BMI microswatch assay described. Theperformance index is determined by comparing the performance of thevariant with that of the enzyme of SEQ ID NO:2, with the enzyme dosagerange being 0.1-5 ppm in all cases. Enzymes having a performance indexof 1.1 or greater are viewed to be cold water proteases.

B. Assays

TCA Assay for Protein Content Determination

B. subtilis cultures were grown 2-3 days at 37° C., shaking at 250-300rpm with humidified aeration. The cells were removed from theenzyme-containing culture supernatant, by centrifugation and/orfiltration. The protease/protein/enzyme concentration was determinedusing a TCA precipitation assay. An aliquot (20-25 ul) of culturesupernatant was transferred to a 96-well flat bottom microtiter plate(MTP; Costar 9017 medium binding clear polystyrene plate) containing 100μL/well of 0.25 N HCl. The “baseline” read was determined by lightscattering/absorbance reading at 405 nm following 5 seconds of mixing.100 μL/well of 30% (w/v) trichloroacetic acid (TCA) was added to theHCl-containing plate and incubated for 10 minutes at room temperature tofacilitate protein precipitation. The light scattering/absorbance at 405nm of this “test” plate was determined after 5 seconds of mixing. Theturbidity/light scattering increase in the samples correlates to thetotal amount of precipitable protein in the culture supernatant. Thecalculations were performed by subtracting the “baseline” reading(obtained after addition of HCl) from the “test” reading (obtained afteraddition of TCA) to provide a relative measure of total protein present.A conversion factor relating protein precipitation with proteinconcentration was determined for a GG36 standard of known concentration.This conversion factor can be used for all variants since precipitationis linear with concentration. If desired, a standard curve can becreated by calibrating the TCA readings with AAPF protease assays (seebelow) of clones with known specific activity. However, the TCA resultsare linear with respect to protein concentration from 50 to 500 partsper million (ppm) of protein (where 1 ppm corresponds to 1 mg/L) and canthus be plotted directly against enzyme performance for the purpose ofchoosing variants with desired performance.

AAPF Protease Assay

In order to determine the protease activity of the serine proteasevariants, more specifically subtilisin variants, the hydrolysis ofN-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenyl-p-nitroanilide(suc-AAPF-pNA) was measured. The reagent solutions used were: 100 mMTris/HCl, pH 8.6, containing 0.005% TWEEN®-80 (Tris dilution buffer);100 mM Tris buffer, pH 8.6, containing 1 mM CaCl, and 0.005% TWEEN®-80(Tris/Ca buffer); and 160 mM suc-AAPF-pNA in DMSO (suc-AAPF-pNA stocksolution) (Sigma: S-7388). To prepare a suc-AAPF-pNA working solution, 1ml suc-AAPF-pNA stock solution was added to 100 ml Tris/Ca buffer andmixed well for at least 10 seconds. The assay was performed by adding 10μA of diluted protease solution to each well of a 96-well MTP,immediately followed by the addition of 190 μl of 1 mg/ml suc-AAPF-pNAworking solution. The solutions were mixed for 5 sec, and the absorbancechange in kinetic mode (25 readings in 5 minutes) was read at 405 nm inan MTP reader, at 25° C. The protease activity was expressed as AU(activity=ΔOD·min⁻¹ ml⁻¹).

BMI Microswatch Assay (BMI Assay)

Pre-rinsed and punched blood, milk and ink (BMI) stained microswatches(EMPA116) of 5.5 millimeter circular diameter in 96 well microtiterplates (MTP; Corning 3641) were obtained from Center for Test materialsBV (Vlaardingen, The Netherlands).

Detergents in Table 1 were prepared by mixing for at least 30 minuteswith the appropriate level of water hardness (3:1Ca:Mg.—CaCl₂:MgCl₂.6H₂O) in Milli-Q water for detergents compositions104 and 105, and in 2 mM sodium carbonate buffer pH 10.3 for detergentcompositions 101, 102, 103, 106 and 107 as described in Table 1. Thedetergents were centrifuged and filtered to remove precipitate andchilled on ice for 30 minutes prior to use for assays carried out at 16°C.

Enzyme concentrations were equalized to a desired fixed concentrationranging from 20-50 ppm relative to a standard of purified GG36. Thespecific activity of GG36 using AAPF as a substrate was used to convertbaseline subtracted TCA values into enzyme concentration in ppm. Onceenzyme concentration was determined in ppm, a simple formula was used tocalculate the volume of each variant required to add to a fixed volumeof buffer (300-600 μL) in order to achieve the desired stock enzymeconcentration:x=(target ppm)(v _(b))/(y-target ppm)

Where x=volume enzyme, y=enzyme concentration, v_(b)=buffer volume

A Perkin-Elmer Janus robot with a Versispan 8 channel arm was used todispense variable volumes of enzyme from the source plate (Axygen halfdeep well plate with pooled harvested variants used in the TCA enzymeconcentration assay) into the buffer-filled destination plate usingconductive tips. Samples were mixed three times by pipetting up anddown. The accuracy of the enzyme dilutions was validated by measuringthe AAPF activity of the equalized plate and comparing it to that of thesource plate, to verify that the correct dilutions had been made.

After equalization, 5-15 μL of enzyme solution was added to adetergent-filled BMI microswatch plate to reach a final volume of ˜200μL. In some instances, the enzyme samples were not equalized, and wereinstead all diluted equally from the stock plate to give a working rangeof 0.1 to 5 ppm. Optimal target concentrations for each assay weredetermined from a dose response curve measuring cleaning activity overthis range for a given detergent.

The MTP was sealed with foil (Bio-Rad) and incubated in iEMSincubator/shaker (Thermo/Labsystems) pre-set to 16° C. in a cold roomset to 4° C. or at 25° C. on the benchtop for 15-30 minutes at 1400 rpm.Following incubation, 120 μL of supernatant was transferred to a freshMTP (Corning 9017) and read at 600 nm using the SpectraMax reader. Trueabsorbance readings were obtained by subtracting a blank control (noenzyme) from each value.

A performance index (PI) was calculated for each variant. Theperformance index is the ratio of the absorbance of the supernatantproduced by variant enzyme cleaning to the absorbance produced by GG36cleaning at a fixed enzyme concentration. PI values were calculated bydividing the absorbance of a variant by that of the control on a givenplate. If multiple versions of the same variant were obtained in alibrary screen, their PI values were averaged to derive a singlerepresentative value. A performance index (PI) that is greater than 1(PI>1) indicates superior cleaning by a variant as compared to thestandard (e.g., GG36), while a PI of 1 (PI=1) identifies a variant thatperforms the same as the standard, and a PI that is less than 1 (PI<1)identifies a variant that performs worse than the standard.

TABLE 1-1 Detergent Compositions Detergent Compositions 101 102 103 104105 106 107 Sodium C₁₂₋₁₇ alkyl sulfate 0.8 5.88 6.93 6.93 Sodium linearalkylbenzenesulfonate with aliphatic 14.47 13.6 8.98 8.2 7.02 11.5 8.78chain length C₁₁-C₁₂ Sodium C₁₂₋₁₇ alcohol ethoxy sulfate 2 1.28 1.511.51 1.12 C₁₀₋₁₅ alcohol ethoxylate with average 7 moles 0.06 0.12 1.71.33 ethoxylation Zeolite 2.7 15.21 17.78 20.54 2.04 1.57 SodiumSilicate 5.7 8 3.72 3.44 2.94 4.76 5.87 Citric Acid 0 0.5 0 1.08 1Sodium tripolyphosphate Sodium Carbonate 11.93 13.47 26.09 29.7 28 23.2823.77 Nonanoyloxybenzenesuplhonate 5.77 2.74 0.63 Oxaziridinium-basedbleach booster 0.04 0 0 0.01 0.01 TetrasodiumS,S,-ethylenediaminedisuccinate 0.26 0 Diethylenetriamine penta(methylene phosphonic 0.61 0.49 1.6 acid), heptasodium salt (DTPA)Hydroxyethane dimethylene phosphonic acid 0.47 0.55 Ethylene diaminetetraacetate MgSO4 0.05 0 0 0.81 2 Sodium Percarbonate 1.53 7.05 6.5310.4 19.35 14.11 Tetra Acetyl Ethylene Diamine 1.15 4.51 3.26 SodiumPerborate Monohydrate Carboxymethyl cellulose 0.21 0.21 0.38 0 0 1.010.91 (e.g. Finnfix BDA ex CPKelco) Sodium acrylic acid/acrylic-maleicacid copolymer 1.55 0.75 3.79 3.48 2.98 1.84 1.75 Polyethyleneglycol/vinyl acetate random graft copolymer 0.3 0.6 0.96 0.91Photobleach- zinc phthalocyanine tetrasulfonate 0.0066 0.0022 0.0012Fluorescent Brightener 0.2 0.13 0.11 0.11 0.11 0.11 0.11 Suds suppressorgranule 0.01 0.16 0.01 0.05 0.05 Miscellaneous (Dyes, perfumes, processaids, Balance Balance Balance Balance Balance Balance Balance moistureand sodium sulphate)

TABLE 1-2 Final Detergent, Water Hardness, and Buffer ConcentrationsUsed for BMI Microswatch Assays Final Detergent Final Sodium DetergentConcentration Final Water Carbonate Buffer Composition (g/L) Hardness*(gpg) Concentration (mM) 1 1.2 12 2 2 2.25 12 2 3 0.719 6 2 4 0.625 6 05 0.625 6 0 6 7.69 20 2 7 7.69 20 2 *(3:1 Ca:Mg) Concentration asdetailed in text.Performance Index

The performance index compares the performance of the variant (measuredvalue) and the standard enzyme (theoretical value) at the same proteinconcentration. In addition, the theoretical values can be calculated,using the parameters of a performance dose response curve of thestandard protease

Example 2 Construction of Variants and Combinatorial Libraries of GG36

This Example describes the cold water cleaning of GG36 variants andlibraries constructed in B. subtilis using the pHPLT-GG36 B. subtilisexpression plasmid. This B. subtilis expression plasmid contains theGG36 expression cassette shown below, the B. licheniformis LAT promoter(Plat), and additional elements from pUB110 (McKenzie et al., Plasmid,15:93-103, 1986) including a replicase gene (reppUB), aneomycin/kanamycin resistance gene (neo) and a bleomycin resistancemarker (bleo) (FIG. 4 in U.S. Pat. No. 6,566,112). The pHPLT-GG36plasmid map was provided in WO2011140364. The GG36 expression cassettesequence is provided below.

The DNA sequence of GG36 (the signal sequence is shown in lower caseletters, propeptide in lower case, underlined text, and GG36 maturesequence in uppercase letters) is provided below:

(SEQ ID NO: 3) gtgagaagcaaaaaattgtggatcgtcgcgtcgaccgcactactcatttctgttgctttcagttcatcgatcgcatcggctgctgaagaagcaaaagaaaaatatttaattggctttaatgagcaggaagctgtcagtgagtttgtagaacaagtagaggcaaatgacgaggtcgccattctctctgaggaagaggaagtcgaaattgaattgcttcatgaatttgaaacgattcctgttttatccgttgagttaagcccagaagatgtggacgcgcttgagctcgatccagcgatttcttatattgaagaggatgcagaagtaacgacattgGCGCAATCAGTGCCATGGGGAATTAGCCGTGTGCAAGCCCCAGCTGCCCATAACCGTGGATTGACAGGTTCTGGTGTAAAAGTTGCTGTCCTCGATACAGGTATTTCCACTCATCCAGACTTAAATATTCGTGGTGGCGCTAGCTTTGTACCAGGGGAACCATCCACTCAAGATGGGAATGGGCATGGCACGCATGTGGCCGGGACGATTGCTGCTTTAAACAATTCGATTGGCGTTCTTGGCGTAGCGCCGAGCGCGGAACTATACGCTGTTAAAGTATTAGGGGCGAGCGGTTCAGGTTCGGTCAGCTCGATTGCCCAAGGATTGGAATGGGCAGGGAACAATGGCATGCACGTTGCTAATTTGAGTTTAGGAAGCCCTTCGCCAAGTGCCACACTTGAGCAAGCTGTTAATAGCGCGACTTCTAGAGGCGTTCTTGTTGTAGCGGCATCTGGAAATTCAGGTGCAGGCTCAATCAGCTATCCGGCCCGTTATGCGAACGCAATGGCAGTCGGAGCTACTGACCAAAACAACAACCGCGCCAGCTTTTCACAGTATGGCGCAGGGCTTGACATTGTCGCACCAGGTGTAAACGTGCAGAGCACATACCCAGGTTCAACGTATGCCAGCTTAAACGGTACATCGATGGCTACTCCTCATGTTGCAGGTGCAGCAGCCCTTGTTAAACAAAAGAACCCATCTTGGTCCAATGTACAAATCCGCAATCATCTAAAGAATACGGCAACGAGCTTAGGAAGCACGAACTTGTATGGAAGCGGACTTGTCAATGCAGAAGCTGCAACTCGTTAA

The protein sequence of GG36 (the signal sequence is shown in lower caseletters, propeptide in lower case, underlined text, and GG36 matureprotease sequence in uppercase letters) is provided below:

(SEQ ID NO: 4) vrskklwivastallisvafsssiasaaeeakekyligfneqeaysefveqveandevailseeeeveiellhefetipvlsvelspedvdaleldpaisyieedaevttmAQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR.

The DNA libraries and variants were created by extension PCR(WO2011140364), QuickChange mutagenesis (Stratagene), or they weresynthesized at DNA2.0, Inc. or GeneArt. The pHPLT GG36 plasmid was usedfor cloning of the GG36 variant genes or for the mutagenesis reactions.For efficient transformation of the libraries and variants in B.subtilis, 1 microliter of the ligated library products or mutagenesisreactions was amplified using rolling circle amplification by IllustraTempliphi kit according to the manufacturer's instructions (GEHealthcare) to generate multimeric DNA for transformation into Bacillussubtilis. Products of the rolling circle amplification were diluted100-times and used to transform B. subtilis cells (genotype: ΔaprE,ΔnprE, amyE::xylRPxylAcomK-phleo). An aliquot of the transformation mixwas plated on LB plates containing 1.6% skim milk and 10 μg/mL neomycinand incubated overnight at 37° C. Subsequently, the colonies with haloswere inoculated in 120 μl of Luria broth medium containing 10 μg/mLneomycin for plasmid DNA extraction (QIAprep Spin Miniprep kit, Qiagen).The extracted plasmids were sequenced to confirm the presence of thedesired mutations. The variants were expressed in B. subtilis cells(genotype: ΔaprE, ΔprE, amyE::xylRPxylAcomK-phleo) as described inExample 1 (TCA assay), and were further characterized using the BMImicroswatch cleaning assay as described in Example 1. In the followingtables, the detergent compositions (“Detergents”) correspond to thoseshown in Table 1 above. Also, as indicated, the amino acid position islisted according to BPN′ numbering.

TABLE 2-1 BMI cleaning performance of Group D1 variants. PI =Performance Index PI > or = 3 is +++; PI between 2.9 and 2 = ++; PIbetween 1.9 and 1.1 = + in a Detergent Composition as described in Table1 PI Relative to GG36; Sequence of GG36 Variants (BPN′ numbering) BMIassay at 25° C. G020R-N062E-S078G-G118S-S188D-N248D-H249R +++S024R-N062E-G118R-A158E-S188D +++ T022A-S024R-T033S-G118R-S166D-S188D+++ S024R-N062E-S078G-G118S-S188D-Q245R-N248D +++G020R-S024R-N062E-S078D-G118S-P129E-G159D +++G020R-S024R-N062E-S078G-G118D-P129E +++G020R-S024R-N062E-S078G-G118S-P129E-G159D-S188D-Q245R- +++ N248DG020R-N062E-S078G-G118S-G159D-Q245R-N248D +++ S024R-N116L-A158E-S166D+++ G020R-N062E-S078G-G118S-G159D-S188D-H249R +++G020R-S024R-S078G-G118D-P129E-G159D-S188D +++ S024R-G118R-S166D +++T022A-S024R-T033S-G118R-A158E-S166D-A273V +++G020R-N062E-S078G-G118S-P129E-G159D-S188D-Q245R +++T022A-G118R-A158E-S166D +++G020R-S024R-N062E-S078D-G118S-G159D-S188D-Q245R-N248D +++T022A-S024R-G118R-S166D-S188D +++ N062E-S078G-G118S-G159D +++T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248E- +++ E271HG020R-S024R-N062E-S078G-G118D-Q245R-N248D +++S024R-T033S-N116L-A158E-S166D +++S024R-N062E-S078G-G118D-P129E-G159D-Q245R +++G020R-N062E-S078D-G118S-Q245R +++ T022A-S024R-N062E-N116L-A158E +++G020R-N062E-S078G-G118D-P129E +++ S024R-T033S-N062E-N116L-G118R-S188D+++ G020R-N062E-S078G-G118D-P129E-G159D-S188D-H249R +++S024R-S078G-G118S-P129E-G159D-Q245R-N248D +++G020R-N062E-S078G-G118D-S188D-H249R +++T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245T-N248D- +++ E271TS078G-G118S-P129E-G159D-S188D-Q245R-N248D-H249R +++T022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245T-N248H- +++ E271TG020R-S024R-S078D-G118S-G159D-S188D-H249R +++G020R-S078D-G118S-P129E-G159D-Q245R-N248D-H249R +++S024R-N062E-N116L-G118R +++ T022A-S024R-N116L-G118R-A158E-S188D +++S024R-N062E-S078G-G118S-P129E-Q245R +++ S024R-N062E-G118R-A158E +++G020R-N062E-S078G-G118S-G159D-S188D +++G020R-S024R-S078D-G118S-G159D-S188D-Q245R-N248D-H249R +++S024R-S078D-G118S-P129E-G159D-Q245R +++ A098Q-S099T-G102A-S103G +++T022A-G118R-S166D-S188D +++ S024R-S078D-G118S-G159D-S188D-H249R +++T022A-S024R-N116L-G118R-S166D-S188D +++ T022A-N062E-G118R-A158E +++G020R-S078G-G118D-G159D-S188D-Q245R +++S024R-N062E-S078G-G118D-G159D-S188D-Q245R +++T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248E- +++ E271FT022A-S101G-S103A-V104I-G159D-S188E-A232S-Q245R-N248E- +++ E271FG020R-N062E-S078G-G118S-P129E-N248D-H249R +++S024R-N116L-G118R-S128I-S166D-S188D +++G020R-S024R-N062E-S078G-G118D-P129E-G159D-S188D-H249R +++G020R-S024R-N062E-S078G-G118D-G159D-S188D-N248D +++G020R-S024R-S078G-G118D-P129E-S188D-Q245R-N248D +++S024R-N062E-S078D-G118S-S188D-H249R +++ T033S-N062E-G118R +++T022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245T-N248E- +++ E271HT033S-G118R-A158E-S166D +++T022Q-S101G-S103A-V104I-G159D-S188E-A232S-Q245V-N248H +++G020R-S024R-S078D-G118D-S188D-Q245R-N248D-H249R +++ T022A-S024R-N062E+++ G020R-S024R-N062E-S078D-G118D-G159D-S188D-R247L +++T022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245R-N248E- +++ E271TG020R-N062E-S078G-G118S-P129E-G159D-Q245R-N248D +++T022Q-S101G-S103A-V104I-G159D-S188E-A232S-Q245R-N248H +++P014S-G020R-S024R-S078G-G118S-P129E-S188D-Q245R-N248D +++T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245R-N248E- +++ E271LG020R-S078D-G118D-G159D-S188D-H249R +++T022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245R-N248D- +++ E271TT022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245T-N248E- +++ E271LS024R-N062E-S078D-G118S-S188D-Q245R +++T022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245R-N248E- +++ E271HG020R-S078D-G118S-P129E-G159D-H249R +++T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245R-N248H- +++ E271FT022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245R-N248H- +++ E271HS024F-T033S-S101G-S103A-V104I-N116A-Y209A-T213A-A232V +++S024F-S101G-S103A-V104I-N116L-S128N-Y209V-G211Q-T213A- +++ A232VS024R-S078G-G118S-G159D-S188D-Q245R-N248D +++G020R-S024R-S078G-G118S-S188D-Q245R +++G020R-S024R-N062E-S078D-G118D-P129E-G159D-Q245R +++S024R-S078D-G118D-G159D-S188D-H249R +++G020R-S078G-G118S-G159D-S188D-H249R +++G020R-S024R-N062E-S078G-G118S-S188D-N248D +++ S078D-G118S-G159D-Q245R+++ G020R-S024R-N062E-S078G-G118D-P129E-G159D-N248D-H249R +++S024R-N062E-S078G-G118S-P129E-Q245R-N248D +++G020R-S078G-G118S-P129E-S188D-Q245R-N248D +++S024R-N062E-S078G-G118S-G159D-Q245R +++G020R-N062E-S078D-G118D-P129E-Q245R +++T022A-S101G-S103A-V104I-G159D-S188E-P210L-A232T-Q245T- +++ N248D-E271TG020R-S024R-N062E-S078G-G118S-P129E +++G020R-S024R-N062E-S078G-G118D-G159D-S188D-H249R +++T022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245V-N248D- +++ E271LA016S-T022A-S101G-S103A-V104I-N116L-S128N-Y209V-A232V +++T022A-S101G-S103A-V104I-G159D-S188E-A232S-Q245T-N248H- +++ E271HG020R-N062E-S078G-G118D-P129E-G159D-H249R +++T022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245R-N248E +++G020R-S024R-S078G-G118D-G159D-S188D-Q245R +++S024R-N062E-S078G-G118S-G159D-Q245R-N248D +++G020R-S078G-G118S-G159D-S188D-Q245R +++S024R-N062E-S078G-G118S-G159D-S188D +++T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245R-N248H- +++ E271HG020R-S024R-N062E-S078G-G118D-G159D +++T022A-S101G-S103A-V104I-G159D-S188D-A232T-V244A-Q245R- +++ N248H-E271HS024R-S078G-G118S-P129E-G159D-S188D-Q245R +++T022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245V-N248D- +++ E271FG020R-S024R-N062E-S078G-G118D-G159D-S188D-Q245R +++T022A-S024R-N062E-S128I-A158E +++ G020R-S024R-S078G-G118D-N248D-H249R+++ S024R-S078G-G118D-P129E-Q245R-H249R +++T022A-S101G-S103A-V104I-G159D-S188D-A232T-Q236K-Q245T- +++ N248EN062E-S078G-G118S-G159D-Q245R-N248D +++ T033S-G118R-S166D +++G020R-S024R-S078G-G118D-P129E-S188D-N248D +++G020R-S078G-G118S-P129E-G159D-S188D-Q245R-N248D +++A016S-T022A-S101G-S103A-V104I-L111V +++T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248E- +++ E271HS024R-N116L-G118R-S128I-A158E-S188D-V199I +++G020R-S024R-S078D-G118D-G159D-H249R +++T022A-S101G-S103A-V104I-G159D-S188E-A232S-Q245R-N248E- +++ E271TG020R-S024R-S078D-G118S-S188D-N248D-H249R +++T022A-S101G-S103A-V104I-G159D-S188D-A232T-Q245R-N248D- +++ E271TG020R-S024R-S078G-G118D-P129E-S188D-H249R +++S024R-N062E-S078G-G118S-S188D-N248D +++T022A-S101G-S103A-V104I-N116L-S128N-Y209A-G211Q-T213A- +++ A232VT022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245R-N248D- +++ E271LS024R-A048V-S078G-G118D-G159D-S188D-H249R +++G020R-S024R-N062E-S078D-G118D +++ G102A-S103G-V104I +++G020R-S024R-S078D-G118D-G159D-Q245R-N248D-H249R +++T022A-T033S-G118R-A158E +++ T022A-S024R-N116L-S128I-S188D +++G020R-S024R-S078D-G118D-P129E-G159D-Q245R-N248D-H249R +++T022Q-S101G-S103A-V104I-G159D-S188E-A232T-Q245T-N248E- +++ E271FS024R-N062E-S078G-G118S-S188D-Q245R +++ S024R-N062E-S078G-G118D-G159D+++ G020R-S024R-N062E-S078D-G118S-P129E-G159D-Q245R +++T022Q-S101G-S103A-V104I-G159D-S188E-A232S-Q245V-N248E- +++ E271TG020R-S078G-G118D-S188D-N248D-H249R +++T022A-S024R-N062E-N116L-G118R-A158E-S166D-S188D +++S078G-G118S-G159D-S188D-H249R +++T022Q-S101G-S103A-V104I-G159D-S188E-A232T-Q245V-N248H- +++ E271FG020R-S078G-G118S-P129E-G159D-S240E-N248D-H249R +++S024F-T033S-S101G-S103A-V104I-N116A-Y209A-G211Q-T213A- +++ A232VN062E-S078D-G118S-S188D-H249R +++T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245V-N248D- +++ E271TG020R-S078G-G118S-G159D-S188D-Q245R-N248D +++T022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245R-N248D- +++ E271FS024R-S078G-G118D-G159D-S188D-Q245R-N248D-H249R +++G020R-S024R-N062E-S078D-G118D-Q245R +++S024R-S078G-G118D-P129E-S188D-Q245R +++N062E-S078G-G118S-S188D-Q245R-N248D-H249R +++S024R-N062E-S078G-G118D-G159D-H249R +++G020R-S078G-G118S-P129E-G159D-S188D-H249R +++S024R-N062E-S078G-G118S-P129E-S188D-H249R +++T022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245R-N248H +++T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245V-N248H- +++ E271LG020R-S101G-S103A-V104I-N116A-S128N-Y209V-G211Q-T213A- +++ A232VS024R-N062E-S078D-G118S-P129E-G159D-H249R +++T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245R-N248D- +++ E271HG020R-N062E-S078G-G118D-P129E-S188D-H249R +++T022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245V-N248E- +++ E271LG020K-T022L-S078G-G118S-P129E-G159D +++ S078G-G118S-S188D-Q245R-N248D+++ T022A-S024F-S101G-S103A-V104I-N116A-S128N-Y209A-A232V +++G020R-S024R-N062E-S078G-G118S-G159D-N248D +++ N062E-G118R-S166D-S188D+++ T022Q-S101G-S103A-V104I-G159D-S188E-A232T-Q245R-N248E- +++ E271TT022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245T-N248H- +++ E271FG020R-N062E-S078G-G118S-G159D-H249R +++ S024R-N062E-N116L-A158E +++S024R-S078G-G118D-P129E-Q245R-N248D-H249R +++G020R-S024R-N062E-S078D-G118S-P129E-S188D-Q245R +++S024R-S078G-G118D-P129E-G159D-S188D-H249R +++T022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245R-N248D- +++ E271HS024R-S078D-G118D-P129E-S188D-Q245R-N248D-H249R +++G020R-S024R-S078G-G118D-P129E-G159D-N248D-H249R +++T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245T-N248E- +++ E271FT022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245T-N248H- +++ E271TS024R-S078D-G118S-P129E-G159D +++G020R-N062E-S078G-G118S-P129E-S188D-Q245R +++T022A-S024R-N116L-G118R-S128I-S188D +++T022A-S101G-S103A-V104I-G159D-S188E-A232L-Q245T-N248E- +++ E271HG020R-S024R-S078D-G118S-P129E-G159D-S188D-Q245R-N248D +++G020R-S024R-N062E-S078G-G118S-P129E-S188D-N248D-H249R +++T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245V-N248H- +++ E271HG020R-S078G-G118D-P129E-G159D-S188D-H249R +++G020R-S024R-S078D-G118S-S188D-Q245R-H249R +++T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248E- +++ E271FT022A-S101G-S103A-V104I-G159D-S188D-A232T-Q245R-N248D- +++ E271FS024R-N062E-S078G-G118S-S188D-N248D-H249R +++G020R-N062E-S078G-G118S-G159D-Q245R +++S024R-N062E-G118R-A158E-S166D-S188D +++S024R-N062E-S078G-G118S-P129E-G159D-N248D-H249R +++G020R-S024R-N062E-S078G-G118S-P129E-A158T-G159D-Q245R- +++ N248DT022Q-S101G-S103A-V104I-G159D-S188E-A232T-Q245R-N248E- +++ E271LG020R-S024R-N062E-S078D-G118S-S188D-H249R +++S024R-S078G-G118S-S188D-N248D +++T022A-S101G-S103A-V104I-G159D-S188E-A232L-Q245V-N248E- +++ E271HT022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245T-N248H- +++ E271HS099G-S101G +++ T022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245V-N248D-+++ E271F G020R-N062E-S078D-G118S-P129E-G159D-Q245R +++T022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245T-N248H- +++ E271HT022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245V-N248H- +++ E271LT022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248E- +++ E271TT022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245T-N248E- +++ E271TT022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245V-N248E- +++ E271HT022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245R-N248H- +++ E271LG020R-S078G-G118S-P129E-G159D-S188D-Q245R-N248D-H249R +++G020R-S024R-N062E-S078G-G118D-S188D-N248D +++S024R-N062E-S078G-G118S-P129E-G159D +++A016S-T022A-S101G-S103A-V104I-N116A-S128N-Y209V-G211Q- +++ A232VS024R-S078G-G118D-S188D-Q245R-N248D-H249R +++G020R-S024R-S078D-G118D-G159D-S188D-Q245R-N248D-H249R +++A016S-T022A-S101G-S103A-V104I-N116A-Y209V-G211Q-T213A- +++ A232VS024R-N062E-S078G-G118D-S188D-H249R +++T022L-S078N-G118R-S166D-T213A-L217E +++T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245R-N248E- +++ E271HS024R-S078G-G118D-G159D-N248D-H249R +++S024R-S078G-G118D-P129E-S188D-Q245R-N248D +++T022Q-S101G-S103A-V104I-G159D-S188E-A232T-Q245T-N248H- +++ E271TT022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245R-N248H- +++ E271HG020R-S024R-S078G-G118S-G159D-S188D-Q245R-N248D +++T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D- +++ E271LS024R-N062E-S078G-G118D-P129E-G159D-H249R +++T022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245T-N248H- +++ E271FG020R-S024R-N062E-S078G-G118S-P129E-G159D-S188D-N248D- +++ H249RT022Q-S101G-S103A-V104I-G159D-S188E-A232T-Q245R-N248H- +++ E271TT022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245R-N248E- +++ E271FG020R-S024R-S078D-G118S-P129E-G159D-H249R +++S024R-N062E-S078G-G118S-P129E-G159D-S188D-Q245R +++T022A-S101G-S103A-V104I-G159D-S188E-A232S-Q245R-N248D- +++ E271HG020R-S024R-S078D-G118S-S188D +++T022A-S101G-S103A-V104I-G159D-S188D-A232T-Q245R-N248E +++T022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245R-N248D- +++ E271HT022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245R-N248H- +++ E271FT022Q-S101G-S103A-V104I-G159D-S188E-A232S-Q245T-N248H- +++ E271TT022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245T-N248H- +++ E271TN062E-S078G-G118D-G159D-N248D-H249R +++ G020R-S078G-G118S-S188D-N248D+++ S024R-S078D-G118S-P129E-S188D +++G020R-S078G-G118D-P129E-S188D-H249R +++G020R-N062E-S078D-G118D-G159D-S188D-H249R +++T022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245T-N248D- +++ E271LT022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245V-N248H- +++ E271TG020R-N062E-S078G-G118S-P129E-G159D-Q245R +++A001E-G020R-S024R-S078G-G118D-G159D-S188D +++T022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245T-N248E- +++ E271LG020R-S078D-G118S-G159D-N248D-H249R +++T022Q-S101G-S103A-V104I-G159D-S188E-A232T-Q245V-N248E- +++ E271LG020R-N062E-S078D-G118S-P129E-H249R +++T022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245R-N248E- +++ E271FS024F-T033S-S101G-S103A-V104I-N116A-Y209V-A232V +++S024R-N062E-N116L-G118R-S128I-A158E +++S024R-S078G-G118D-P129E-G159D-S188D-Q245R +++T022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245V-N248D- +++ E271LT022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245V-N248E- +++ E271HS024R-N062E-S078G-G118S-G159D-N248D +++G020R-S078G-G118S-P129E-S188D-H249R +++S024R-S078G-G118S-P129E-S188D-Q245R +++S024R-N062E-S078D-G118D-G159D-H249R +++T022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245T-N248H- +++ E271HS024R-S078G-G118S-P129E-Q245R-N248D +++ S024R-T033S-G118R-A158E +++G020R-S024R-S078D-G118S-S188D-Q245R-N248D +++G020R-N062E-S078G-G118S-P129E-S188D-Q245R-N248D +++T022A-S101G-S103A-V104I-G159D-S188E-A232S-Q245R-N248D- +++ E271TT022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245R-N248H- +++ E271TT022A-S101G-S103A-V104I-G159D-S188E-A232L-Q245T-N248D- +++ E271HG020R-S024R-N062E-S078G-G118D-P129E-S188D-Q245R-N248D +++G020R-S024R-S078G-G118D-P129E-G159D-S188D-Q245R +++T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245R-N248D- +++ E271HS024R-S078D-G118D-G159D-S188D-Q245R +++G020R-S024R-S078G-G118D-P129E-S188D-N248D-H249R +++S024R-S078G-G118S-G159D-S188D-Q245R-N248D-H249R +++G020R-N062E-S078G-G118S-P129E +++T022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245V-N248D +++T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245R-N248D- +++ E271LA016S-T022A-S024F-S101G-S103A-V104I-N116L-Y209A-G211Q- +++ T213A-A232VT022A-S024R-T033S-S188D +++ G020R-S024R-S078D-G118S-G159D-Q245R-N248D+++ G020R-S024R-N062E-S078G-G118D-G159D-H249R +++G020R-S024R-S078G-G118D-P129E-G159D-S188D-Q245R-N248D +++T022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245T-N248D- +++ E271HN062E-S078G-G118S-N248D +++ T022A-S024R-N062E-S128I +++G020R-S024R-N062E-S078D-G118D-G159D-S188D-Q245R-N248D +++G020R-S024R-N062E-S078G-G118D-P129E-G159D-H249R +++G020R-S024R-S078D-G118D-S188D-N248D-H249R +++S024R-S078G-G118D-G159D-S188D +++S024F-S101G-S103A-V104I-N116A-Y209A-G211Q-A232V +++T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245R-N248E- +++ E271LT022A-S024R-T033S-N062E-G118R-S188D +++G020R-S078D-G118D-P129E-G159D-S188D-Q245R-H249R +++T022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245R-N248D- +++ T255M-E271TS024R-N062E-S078D-G118S-N248D-H249R +++T022A-S101G-S103A-V104I-G159D-S188E-A232L-Q245R-N248D- +++ E271HG020R-S024R-S078D-G118D-P129E-G159D-S188D-H249R +++T022Q-S101G-S103A-V104I-G159D-S188E-A232S-Q245T-N248D- +++ E271LG020R-S024R-N062E-S078G-G118D-P129E-Q245R-N248D +++T022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245R-N248H- +++ E271FS024R-G118R-S128I-A158E-S188D +++S024R-N062E-S078G-G118S-P129E-G159D-S188D-Q245R-N248D- +++ H249RT022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245V-N248H +++T022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245T-N248E- +++ E271HT022A-G118R-S128I-A158E-S188D +++T022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245R-N248E- +++ E271TS078G-G118S-P129E-G159D-S188D-Q245R-H249R +++S024R-N062E-S078G-G118D-P129E-G159D +++ N116L-G118R-S128I-A158E +++T022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245R-N248H- +++ E271TG020R-S024R-S078G-G118D-P129E-G159D-S188D-N248D-H249R +++G020R-S024R-N062E-S078D-G118S-G159D-Q245R-N248D +++S024R-S078D-G118S-P129E-G159D-Q245R-H249R +++S024R-N062E-S078D-G118D-G159D +++ S024R-T033S-G118R-S188D +++T022A-S101G-S103A-V104I-G159D-S188E-A232L-Q245R-N248H- +++ E271LT022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245V-N248E- +++ E271LS024R-S078G-G118D-P129E-S188D +++ S078G-G118D-G159D-S188D-H249R +++T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245T-N248H- +++ E271HT022A-S101G-S103A-V104I-G159D-S188D-A232T-Q245V-N248D- +++ E271LA016S-T033S-S101G-S103A-V104I-N116A-Y209V-G211Q-T213A- +++ A232VT022A-S024R-N062E-N116L-S128I-A158E +++T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245T-N248H- +++ E271FS024R-G118R-S128I-S188D +++ G020R-S078G-G118D-G159D-Q245R-N248D-H249R+++ N062E-S101A-S103A-V104I-S188D-P210I-A232V-Q245R +++S024R-N062E-S078G-G118S-P129E-G159D-S188D-Q245R-N248D +++S024R-N062E-S078G-G118D-N248D-H249R +++S024R-N062E-S078G-G118S-G159D-S188D-N248D +++S024R-N062E-S078G-G118S-P129E-S188D-Q245R +++ T022A-T033S-G118R-S166D+++ T022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245R-N248E- +++ E271TS078G-G118D-S188D-Q245R-N248D-H249R +++ T022A-S024R-G118R-S128I +++T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245T-N248E- +++ E271LG020R-N062E-S078D-G118S-S188D +++T022A-T033S-S101G-S103A-V104I-N116L-G159D-S188D-T213A- +++A232V-Q245R-N248D-E271FT022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245V-N248H- +++ E271TS024R-S078D-G118S-P129E-G159D-N248D-H249R +++G020R-S078G-G118S-G159D-N248D-H249R +++ G020R-N062E-S078G-G118S-S188D+++ T022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245R-N248D- +++ E271TT022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245T-N248H +++G020R-S024R-N062E-S078D-G118S-P129E-G159D-Q245R-N248D +++G020R-N062E-S078D-G118S-G159D-Q245R +++ S024R-S128I-A158E +++T022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245R-N248H- +++ E271HS024R-N062E-S078D-G118S-S188D-N248D-H249R +++A016S-S024F-S101G-S103A-V104I-N116L-Y209A-L217S-A232V +++S003T-S024R-N062E-S078D-G118S-N248D +++G020R-S024R-N062E-S078D-G118D-S188D +++T022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245V-N248D- +++ E271HT022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245V-N248D- +++ E271HG020R-N062E-S078D-G118S-P129E-S188D-Q245R +++T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245R-N248D- +++ S265G-E271HG020R-S078G-G118D-P129E-G159D-Q245R-H249R +++T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245V-N248E- +++ E271LG020R-S024R-S078G-G118S-G159D-S188D-N248D +++S078G-G118D-G159D-S188D-Q245R-N248D-H249R +++G020R-N062E-S078D-G118S-G159D-Q245R-N248D +++T022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245T-N248H- +++ E271HS024R-N062E-S078D-G118S-P129E-S188D-Q245R-N248D-H249R +++T022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245R-N248E +++T022Q-S101G-S103A-V104I-G159D-S188E-A232T-Q245R-N248D- +++ E271LG020K-T022L-S078N-S166D-T213A-L217E +++G020R-S078D-G118S-P129E-G159D-Q245R +++ T022A-T033S-N116L-G118R-A158E+++ G020R-S024R-S078G-G118S-P129E-G159D-S188D-Q245R +++A001G-T022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245T- +++ N248H-E271LS024R-S078D-G118S-G159D-Q245R +++G020K-T022L-S078N-G118R-S166D-T213A-L217E +++S078G-G118S-P129E-S188D-Q245R +++T022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245T-N248E +++T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245V-N248E- +++ E271F S188D+++ S024R-N062E-S078G-G118D-H249R +++T022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245T-N248H- +++ E271LT022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245V-N248E- +++ E271FG020R-S024R-N062E-S078D-G118S-P129E-S188D +++T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245T-N248H- +++ E271LS024R-S078D-G118D-P129E-S188D-H249R +++T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245V-N248D- +++ E271HG020R-S078G-G118D-G159D +++T022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245R-N248D- +++ E271LT022A-N062E-G118R-S128I-S188D +++ T022A-N116L-G118R-A158E-S166D-S188D+++ S024R-N062E-N116L +++ T022A-S024R-N116L-S128I-S166D-S188D +++T022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245T-N248D- +++ E271FN116L-A158E-S166D +++ G020R-S024R-S078D-G118S-P129E-N248D +++T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245R-N248D- +++ E271TT022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245R-N248E- +++ E271HT022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245R-N248H +++T022A-S024R-N062E-N116L-A158E-S166D +++ G020R-N062E-S078D-G118S-P129E+++ T022A-S101G-S103A-V104I-N116L-H120Q-S128N-Y209A-G211Q- +++T213A-A232V T022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245V-N248E- +++E271F A016S-S024F-S101G-S103A-V104I-N116L-S128N-Y209A-G211Q- +++T213A-A232V T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245T-N248H- +++E271L G020R-S024R-N062E-S078G-G118D-P129E-S188D +++T022Q-S101G-S103A-V104I-G159D-S188E-A232S-Q245R-N248E- +++ E271FN062E-S101A-S103A-V104I-A232V-Q245R +++T022A-S101G-S103A-V104I-G159D-S188E-A232S-Q245R-N248H +++T022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245T-N248E- +++ E271LS024R-N062E-N116L-G118R-S128I +++T022Q-S101G-S103A-V104I-G159D-S188E-A232T-Q245T-N248H- +++ E271FT022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245T-N248E- +++ E271HT022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245V-N248D- +++ E271FT022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245R-N248E- +++ E271FT022A-S101G-S103A-V104I-G159D-S188E-A232S-Q245T-N248E- +++ E271HT022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245R-N248H- +++ E271HN062E-G118R-S128I-A158E +++T022Q-S101G-S103A-V104I-G159D-S188E-A232T-Q245T-N248H- +++ E271HS078D-G118S-G159D-Q245R-H249R +++ S103A-V104I-S128N-L148I +++G020R-S024R-N062E-S078D-G118D-P129E-S188D-H249R +++T022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245R-N248H- +++ E271HS024R-G118R-S188D +++T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245V-N248E- +++ E271FT033S-N062E-G118R-A158E-S188D +++T022A-S024F-S101G-S103A-V104I-N116A-S128N-Y209V-G211Q- +++ T213A-A232VS024R-N062E-S078G-G118S-P129E-G159D-S188D-H249R +++S078G-G118S-P129E-G159D-H249R +++T022A-S101G-S103A-V104I-G159D-S188D-A232T-Q245V-N248E- +++ E271HN062E-N076D-S101A-S103A-V104I-A232V-Q245R +++ G020K-S024F-S078N-L217E+++ S103A-S128N-L148I +++S024F-S101G-S103A-V104I-N116L-Y209A-G211Q-T213A-A232V +++G020R-S024R-S078D-G118S-P129E-G159D-S188D-Q245R-N248D- +++ H249RG020R-N062E-S078D-G118D-P129E-G159D-H249R +++S078G-G118D-P129E-G159D-Q245R-H249R +++G020R-N062E-S078D-G118S-G159D-Q245R-N248D-H249R +++G020R-N062E-S078G-G118S-N248D-H249R +++T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245R-N248E- +++ E271LG020R-S024R-S078D-G118D-G159D-S188D +++ T033S-N116L-A158E +++G020K-S024F-S166D-L217E +++ S024R-S078D-G118D-G159D-S188D-Q245R-H249R+++ G020R-S024R-N062E-S078G-G118S-G159D-N248D-H249R +++T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245T-N248H- +++ E271HT022A-S024R-T033S-G118R-A158E-S166D +++ N062E-S078D-G118D-G159D-Q245R+++ S078G-G118S-P129E-G159D-S188D-H249R +++G020R-N062E-S078G-G118S-P129E-G159D +++T022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245T-N248H- +++ E271FS078D-G118S-G159D-H249R +++ S078G-G118D-P129E-G159D-H249R +++G020R-S024R-S078D-G118D-P129E-S188D-N248D-H249R +++S024R-N062E-S078G-G118S-G159D-S188D-H249R +++G020R-S078D-G118S-G159D-S188D-Q245R +++G020R-N062E-S078D-G118S-G159D-S188D-H249R +++G020R-S024R-N062E-S078G-G118S-P129E-Q245R-N248D +++G020R-N062E-S078G-G118D-S188D-Q245R-N248D +++S024R-N062E-S078D-G118S-G159D-S188D +++G020R-S024R-N062E-S078G-G118S-P129E-Q245R +++G020R-S024R-S078D-G118D-G159D +++G020R-S078G-G118S-G159D-S188D-N248D-H249R +++T022Q-S101G-S103A-V104I-G159D-S188E-A232T-Q245T-N248H +++T022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245R-N248E- +++ E271LT022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248H +++T022A-S024R-N116L-G118R-S128I-S166D-S188D +++S024R-N062E-S078G-G118S-G159D-S188D-Q245R-N248D +++A016S-S024F-S101G-S103A-V104I-N116L-S128N-Y209V-G211Q- +++ A232VT022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245V-N248H- +++ E271LS024R-S078G-G118S-P129E-S188D-Q245R-N248D-H249R +++S024R-S078D-G118D-P129E-Q245R-H249R +++T022Q-S101G-S103A-V104I-G159D-S188E-A232S-Q245V-N248D- +++ E271FG020K-S024F-S078N-G118R-S166D-L217E +++ S024F-G118R-S166D-T213A-L217E+++ G020R-S024R-S078D-G118S-P129E-S188D-Q245R-N248D-H249R +++T022A-S024R-T033S-S078N-S166D-S188D +++ G020R-S024R-S078G-G118D-Q245R+++ G020K-S024F-S078N-G118R-S166D-T213A-L217E +++G020K-T022L-S078G-G118S-G159D-S188D-H249R +++T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245T-N248D- +++ E271LS024R-T033S-G118R-A158E-S166D-S188D +++T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245T-N248D- +++ E271LT022V-S024F-T033S-S101G-S103A-V104I-N116L-Y209A-G211Q- +++ T213A-A232VG020R-S024R-S078G-G118D-G159D-S188D +++ S078N-G118R-S166D-L217E +++S024R-S078G-G118S-G159D-Q245R-N248D +++T022A-S024R-G118R-S128I-A158E-S166D-S188D +++T022A-S101G-S103A-V104I-G159D-S188E-A232L-Q245R-N248H- +++ E271HT022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245T-N248H- +++ E271TG020R-S024R-S078G-G118D-G159D-N248D-H249R +++T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245V-N248D- +++ E271FA016S-T022A-S101G-S103A-V104L-L148I +++G020R-S024R-N062E-S078D-G118S-G159D-N248D +++T022Q-S101G-S103A-V104I-G159D-S188E-A232T-Q245R-N248E- +++ E271HS078G-G118D-P129E-G159D-Q245R +++ S024R-S078G-G118S-S188D-Q245R-N248D+++ G020R-N062E-S078D-G118D-G159D-S188D +++G020R-S024R-N062E-S078D-G118D-G159D-S188D-Q245R +++T022A-T033S-S101G-S103A-V104I-N116L-Y209A-G211Q-T213A- +++ A232VT022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248H- +++ E271HT022A-S024R-T033S-S128I-S188D +++T022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245T-N248D- +++ E271HT022Q-S101G-S103A-V104I-G1159D-S188D-A232L-Q245R-N248E- +++ E271FS024R-S078G-G118S-P129E-S188D-N248D +++G020R-N062E-S078D-G118S-P129E-Q245R +++T022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245T-N248D- +++ E271FT022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245T-N248H +++G020R-S024R-S078D-G118D-G159D-Q245R +++T022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245T-N248E- +++ E271LS101A-N116L-P210I-T213A +++T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248H- +++ E271FG020R-S024R-N062E-S078D-G118D-P129E-Q245R-N248D +++T022A-S024R-N062E-A158E +++T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245T-N248D- +++ E271HG020R-S024R-N062E-S078D-G118D-G159D-S188D-Q245R-N248D- +++ H249RG020R-N062E-S078G-G118S-G159D-S188D-Q245R-N248D +++N062E-S078G-G118S-S188D-H249R +++A016S-S024F-T033S-S101G-S103A-V104I-N116L-Y209V-G211Q- +++ T213A-A232VG020R-S078G-G118D-S188D-H249R +++T022A-S024F-S101G-S103A-V104I-N116L-S128N-Y209A-G211Q- +++ A232VG020R-N062E-S078D-G118S-G159D-N248D-H249R +++T022A-S101G-S103A-V104I-G159D-S188D-A232T-Q245T-N248D- +++ E271HT022A-S024R-N116L-G118R-A158E-S166D-S188D +++G020R-S024R-S078G-G118S-P129E-G159D-S188D-Q245R-N248D- +++ H249RS024R-N062E-N076D-S101A-S103A-V104I-P210I-A232V-Q245R +++N062E-S078D-G118S-G159D-Q245R-N248D-H249R +++G020R-S024R-S078G-G118D-G159D-S188D-N248D-H249R +++T022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245V-N248E- +++ E271TT022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245V-N248E- +++ E271TS024R-S101A-S103A-V104I-S188D-P210I-A232V-Q245R +++S024R-N062E-N116L-G118R-A158E +++ G020R-S078G-G118D-S188D-Q245R +++T022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245V-N248E- +++ E271LT022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245R-N248E- +++ E271HS024R-S166D-S188D +++T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245T-N248D- +++ E271FA016S-T022A-S024F-S101G-S103A-V104I-N116A-Y209V-G211Q- +++ A232VT022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245T-N248D- +++ E271FG020R-N062E-S078G-G118D-G159D-S188D-Q245R-N248D +++T022A-N062E-N116L-G118R-A158E +++ S024R-N062E-N116L-G118R-S128I-S166D+++ T022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245R-N248H +++G020R-S024R-N062E-S078D-G118D-H249R +++T022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245R-N248E- +++ E271LS024R-N062E-G118R-S128I-S188D +++ N062E-S078G-G118S-G159D-N248D-H249R+++ G020K-T022L-N062E-S078D-G118S-G159D-H249R +++N062E-S078G-G118D-Q245R-N248D +++G020R-S024R-N062E-S078D-G118D-G159D-S188D-H249R +++T022A-S024F-S101G-S103A-V104I-N116L-Y209V-T213A-A232V +++T022A-S101G-S103A-V104I-G159D-S188D-A232T-Q245R-N248D- +++ E271LS024R-N116L-G118R-S128I-A158E +++T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245R-N248E- +++ E271FT022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245V-N248E- +++ E271TT022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245V-N248D- +++ E271LA016S-S101G-S103A-V104L-L148I +++T022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245T-N248H- +++ E271TG020R-N062E-S078G-G118S-S188D-Q245R +++T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245V-N248E- +++ E271HS024R-S078D-G118D-S188D-Q245R-N248D-H249R +++G020R-S024R-S078D-G118S-P129E-Q245R-N248D +++G020R-S078G-G118D-G159D-S188D +++T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245T-N248E- +++ E271TG020R-S024R-S078D-G118D-G159D-N248D-H249R +++S024R-N062E-S078G-G118S-P129E-S188D-Q245R-N248D +++T022A-S101G-S103A-V104I-G159D-S188E-A232L-Q245T-N248D- +++ E271TT022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245R-N248E +++T022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245T-N248H- +++ E271FT022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245T-N248E- +++ E271FT022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245R-N248D- +++ E271LG020R-S024R-N062E-S078D-G118S-P129E-N248D +++S024R-N062E-S078D-G118S-P129E-G159D-N248D-H249R +++T022Q-S101G-S103A-V104I-G159D-S188E-S212L-A232S-Q245V- +++ N248D-E271TS024R-S078G-G118D-G159D-H249R +++ T022A-N116L-G118R-S128I-S188D +++T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245R-N248D +++N062E-S078G-G118D-S188D-Q245R +++ G020K-S078N-S166D-L217E +++N062E-S078G-G118S-P129E-S188D-Q245R +++T022Q-S101G-S103A-V104I-G159D-S188E-A232S-Q245V-N248E- +++ E271FN062E-N116L-G118R-S166D-S188D +++ G020R-S024R-S078D-G118S-G159D-N248D+++ T022A-S024F-S101G-S103A-V104I-N116L-S128N-Y209V-A232V +++S078G-G118S-P129E-G159D-Q245R +++T022A-T033S-S101G-S103A-V104I-N116L-Y209V-G211Q-T213A- +++ A232VS078D-G118S-P129E-G159D-S188D-Q245R-N248D-H249R +++S101G-S103A-V104I-N116L-S128N-Y209A-G211Q-T213A-A232V +++G097P-S101A-S103G S024R-N062E-S078D-G118S-S188D +++G020R-S024R-S078D-G118D-P129E-S188D-H249R +++S024R-N062E-S078G-G118S-G159D +++A016S-T022A-T033S-S101G-S103A-V104I-N116A-Y209A-A232V +++T022A-N116L-G118R-S128I-A158E-S166D +++T022Q-S101G-S103A-V104I-G159D-S188E-A232T-Q245R-N248D- +++ E271TS024R-T033S +++ S024R-G118R-S1281 +++T022A-S101G-S103A-V104I-G159D-S188E-A232L-Q245T-N248E- +++ E271LS024R-N062E-A158E +++ S024R-S078D-G118S-P129E +++G020R-S024R-S078D-G118S-P129E-G159D-S188D-H249R +++T022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245V-N248H- +++ E271FT022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245R-N248H- +++ E271FG020R-S024R-N062E-S078G-G118S-G159D +++T022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245V-N248H- +++ E271LS024R-S078D-G118S-P129E-S188D-Q245R-N248D +++N062E-S078G-G118S-S188D-Q245R-N248D +++ N062E-N116L-G118R-S128I-S188D+++ T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245V-N248D- +++ E271FG020R-S024R-S078D-G118S-G159D-S188D-Q245R-N248D +++T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245T-N248D- +++ E271TT022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245V-N248E- +++ E271TT022A-T033S-N116L-G118R-S188D +++G020R-S024R-N062E-S078G-G118S-P129E-S188D-N248D +++G020R-N062E-S078G-G118S-P129E-G159D-N248D +++S078G-G118S-P129E-G159D-S188D-Q245R +++ N062E-S078D-G118S-G159D-H249R+++ T022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245R-N248D +++T022A-S024R-T033S-N062E-N116L +++T022Q-S101G-S103A-V104I-G159D-S188E-A232S-Q245T-N248E- +++ E271TN062E-S078D-G118S-H249R +++ T022A-S024R-N062E-N116L-G118R-A158E +++T022L-S078N-G118R-S166D-L217E +++G020R-S024R-N062E-S078D-G118D-S188D-Q245R-N248D-H249R +++T022A-S101G-S103A-V104I-G159D-S188E-A232S-Q245R-N248H- +++ E271LG020R-S024R-S078G-G118D-P129E-Q245R-N248D-H249R +++T022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245V-N248D- +++ E271LS078D-G118S-P129E-S188D-Q245R +++S024R-S078G-G118D-G159D-S188D-Q245R-H249R +++S078D-G118D-G159D-S188D-Q245R-H249R +++ S024F-T213A-L217E +++S024F-T033S-S101G-S103A-V104I-N116L-S128N-Y209V-G211Q- +++ A232VT022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245T-N248D- +++ E271HT022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245T-N248E- +++ E271TT022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248H- +++ E271HG020R-S078D-G118D-P129E-G159D-Q245R-H249R +++T022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245R-N248D +++T022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245R-N248D- +++ E271TN062E-S078G-G118S-P129E-Q245R +++ S078G-G118S-S188D-Q245R-N248D-H249R+++ S101A-S103G-V104I-L1481 +++T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245R-N248H- +++ E271FT022Q-S101G-S103A-V104I-G159D-S188E-A232T-Q245V-N248H- +++ E271HT022A-N116L-G118R-A158E +++T022A-S024F-T033S-S101G-S103A-V104I-I107V-N116L-S128N- +++Y209A-G211Q-T213A-A232V +++T022Q-S101G-S103A-V104I-G159D-S188E-A232S-Q245R-N248H- +++ E271TG020R-N062E-S078D-G118D-G159D +++ G020R-N062E-S078D-G118S +++S024R-N062E-S078D-G118D-P129E-G159D-Q245R +++T022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245V-N248E- +++ E271TT022A-S101G-S103A-V104I-G159D-S188D-A232T-Q245V-N248D- +++ E271HS078G-G118S-G159D +++ S024F-G118R-T213A-L217E +++S024R-N062E-S078D-G118S-P129E-Q245R-N248D +++G020R-S024R-S078D-G118S-P129E-G159D-Q245R +++S024R-S078D-G118D-G159D-H249R +++S024R-N076D-S101A-S103A-V104I-S188D-P210I-A232V-Q245R +++G020R-S024R-N062E-S078D-G118D-P129E-G159D-H249R +++G020R-S024R-N062E-S078G-G118D-S188D-N248D-H249R +++G020R-S024R-N062E-S078G-G118S-P129E-S188D-H249R +++T022Q-S101G-S103A-V104I-G159D-S188E-A232S-Q245V-N248D +++T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245T-N248E- +++ E271HT022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245V-N248D- +++ E271TG020R-S024R-S078D-G118D-P129E-G159D-N248D-H249R +++G020R-S024R-N062E-S078D-G118S-P129E-G159D-N248D +++N062E-S078G-G118D-P129E-H249R +++ G020K-T022L-S078N-G118R-T213A-L217E+++ G020R-S024R-N062E-S078G-G118D-P129E-S188D-H249R +++T022A-S024R-T033S-N116L-S166D-S188D +++S024R-S078G-G118D-G159D-S188D-H249R +++ T033S-N116L-G118R-A158E-S188D+++ T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245V-N248H +++S101A-S103A-V104I-S188D-P210I-A232V-Q245R +++G020R-S024R-N062E-S078G-G118D-S188D-Q245R +++T022A-S024R-T033S-N062E-N116L-G118R-P129E-S188D +++T022A-S101G-S103A-V104I-G159D-S188E-A232S-Q245T-N248E +++S024R-S078G-G118S +++ N062E-S101A-S103A-V104I-P210I-A232V-Q245R +++N062E-S078G-G118D-G159D +++T022A-S024R-N062E-N116L-G118R-S128I-S166D-S188D +++S024R-S078D-G118D-G159D-Q245R +++G020R-N062E-S078G-G118S-G159D-S188D-N248D-H249R +++T022Q-S101G-S103A-V104I-G159D-S188E-A232S-Q245R-N248E- +++ E271HT022Q-S101G-S103A-V104I-G159D-S188D-A232T-V244I-Q245R- +++ N248H-E271LS024R-S078G-G118S-P129E-G159D-N248D +++S024R-N062E-S078G-G118S-G159D-H249R +++T022A-S024F-T033S-S101G-S103A-V104I-N116L-Y209V-G211Q- +++ A232VA016S-S101G-S103A-V104I-N116A-S128N-Y209V-G211Q-T213A- +++ A232VG020R-N062E-S078G-G118D-S188D +++T022A-S101G-S103A-V104I-G159D-S188E-A232S-Q245V-N248E- +++ E271HT022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245V-N248E- +++ E271LG020K-S166D-L217E +++T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245T-N248E- +++ E271TG020R-N062E-S078D-G118D-Q245R +++S024R-N062E-S078D-G118S-P129E-S188D-Q245R-N248D +++A016S-T022A-S024F-S101G-S103A-V104I-N116A-S128N-Y209A- +++ G211Q-A232VS024F-T033S-S101G-S103A-V104I-N116L-Y209A-A232V +++ T033S-G118R-S188D+++ T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245T-N248E- +++ E271HT022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245T-N248D- +++ E271TG020R-S024R-S078G-G118S-P129E-G159D-S188D-N248D +++T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245T-N248H- +++ E271FG020R-S078G-G118D-P129E-S188D-Q245R +++G020R-S078G-G118D-G159D-S188D-Q245R-N248D +++S024R-S078G-G118D-P129E-N248D-H249R +++ G020R-S024R-S078D-G118D-P129E+++ N062E-S078D-G118S-S188D-Q245R +++G020R-S024R-S078G-G118S-G159D-S188D +++T022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245V-N248H- +++ E271LT022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245R-N248D- +++ E271LN062E-S078G-G118S-G159D-H249R +++ G020R-S078D-G118S-P129E-G159D-N248D+++ G020R-S024R-S078D-G118S-G159D-S188D-Q245R +++G020R-N062E-S078G-G118D-P129E-S188D-Q245R-N248D +++T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245R-N248H +++G020R-S024R-S078D-G118S-G159D-V244L-Q245R-N248D +++G020R-S024R-S078G-G118D-P129E-S188D-Q245R-N248D-H249R +++T022A-S024F-T033S-S101G-S103A-V104I-N116A-Y209A-A232V +++G020R-S024R-N062E-S078D-G118S-P129E-G159D-A272D +++N116L-G118R-S128I-A158E-S166D +++ S024R-S078D-G118S-P129E-H249R +++S024R-N062E-S078G-G118S-P129E-N248D +++G020R-S078G-G118S-P129E-G159D-S188D +++G020R-S024R-S078D-G118S-G159D-N248D-H249R +++S024R-S078D-G118D-G159D-S188D-Q245R-N248D-H249R +++T033S-S101G-S103A-V104I-N116L-Y209A-G211Q-T213A-A232V +++T033S-S101G-S103A-V104I-N116A-Y209A-T213A-A232V +++T022A-S101G-S103A-V104I-G159D-S188E-A232S-Q245V-N248H +++N062E-S078G-G118S-N248D-H249R +++G020R-N062E-S078D-G118D-G159D-S188D-Q245R +++T022A-S024F-S101G-S103A-V104I-N116A-Y209A-T213A-A232V +++N043F-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D- +++ E271FS024R-S078G-G118D-P129E-G159D +++G020R-N062E-S078D-G118D-G159D-Q245R-N248D +++ S024R-S166D +++S024R-S078G-G118S-P129E-N248D +++ A016S +++N076D-S101A-S103A-V104I-S188D-P210I-A232V-Q245R +++ G020K-L217E +++G118R-S166D-S188D +++ S024R-S078D-G118S-G159D-S188D +++S024R-S078D-G118S-P129E-S188D-Q245R +++A016S-S101G-S103A-V104I-N116A-S128N-Y209A-T213A-A232V +++ N116L-S166D+++ S024R-N062E-S078G-G118D-P129E-G159D-S188D-Q245R +++G020R-S024R-S078D-G118D-P129E-N248D-H249R +++N062E-S078G-G118S-G159D-S188D-Q245R +++T022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245V-N248E- +++ E271HS024R-N062E-S078D-G118S-P129E-H249R +++ G118R-S166D-T213A +++G020R-S024R-N062E-S078G-G118S-P129E-G159D +++G020R-S024R-S078D-G115W-G118D-G159D-N248D +++A016S-S024F-S101G-S103A-V104I-N116A-Y209A-A232V +++S024R-N062E-N116L-S128I-A158E +++T022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245V-N248D- +++ E271LS024R-N062E-S078G-G118S-P129E-H249R +++S024R-N062E-S078D-G118D-P129E-N248D-H249R +++G020K-T022L-S024F-S078N-G118R-L217E +++ S101G-G102A-S103A +++G020R-S078G-G118S-P129E-Q245R-N248D +++ G020K-S078N-S166D +++T022A-G118R-A158E +++ G020K-T022L-S024F-S078N-S166D +++T022A-T033S-N062E-N116L-G118R-A158E-S188D +++S024R-S078G-G118S-P129E-G159D-H249R +++S101G-S103A-V104I-N116L-Y209V-G211Q-A232V +++A015D-A016S-S101G-S103A-V104I-N116L-S128N-Y209V-T213A- +++ A232VG020R-N062E-S078D-G118D-P129E-S188D-H249R +++ G020K-S024F-S078N-S128D+++ T033S-N116L-S166D-S188D +++ G020R-S078G-G118S-P129E-G159D +++T022A-S024R-T033S-L082I-G118R-S128I-A158E-S188D +++G020R-S024R-S078D-G118S-S188D-N248D +++G020R-S078G-G118D-P129E-S188D-Q245R-H249R +++ N062E-S078G-G118D-Q245R+++ G020K-T022L-S078N-L217E +++T022A-S101G-S103A-V104I-G159D-S188D-A232T-Q245V-N248E- +++ E271LT022Q-S101G-S103A-V104I-G159D-S188E-A232S-Q245V-N248E- +++ E271HT022Q-S101G-S103A-V104I-G159D-S188E-A232T-Q245T-N248E- +++ E271TA016S-T033S-S101G-S103A-V104I-N116L-Y209V-G211Q-T213A- +++ A232VT022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245K-N248H +++N062E-S078D-G118D-Q245R +++ T022A-S024R-G118R-A158E-S166D +++A016S-S101A-S103A-S128N-L148I +++T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245R-N248E +++S024R-N062E-S078G-G118D +++ S024R-N062E-S078G-G118S-P129E-G159D-S188D+++ S078G-G118D-P129E-G159D-S188D-Q245R-N248D-H249R +++G020R-S078D-G118D-P129E-G159D-Q245R +++T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245V-N248H +++T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D- +++ E271HG020R-S024R-N062E-S078G-G118D-H249R +++ S078D-G118S-P129E-G159D-H249R+++ T022Q-S101G-S103A-V104I-G159D-S188E-A232S-Q245T-N248D- +++ E271HS024R-S078D-G118D-P129E-G159D-S188D-Q245R +++ T022L-T213A-L217E +++G020R-N062E-S078G-G118S-P129E-G159D-S188D-N248D-H249R +++T022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245R-N248H- +++ E271TG020R-N062E-S078G-G118D-Q245R +++G020R-S024R-N062E-S078G-G118D-N248D-H249R +++T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248H- +++ E271TS024R-S078D-G118D-P129E-H249R +++ S024R-N062E-N116L-G118R-S188D +++S024R-N062E-G118R +++T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248E +++G020R-S024R-S078G-G118S-P129E-G159D-S188D-H249R +++T022A-S101G-S103A-V104I-G159D-S188E-A232L-Q245R-N248H +++S024R-S078D-G118S-P129E-G159D-N248D +++T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245V-N248D- +++ E271TG020R-S078G-G118D-P129E-Q245R-N248D-H249R +++G020R-S024R-N062E-S078D-G118D-P129E-S188D-Q245R-N248D +++A016S-S024F-T033S-S101G-S103A-V104I-N116L-Y209V-A232V +++G097P-A098Q-S099G-V104L +++T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245T-N248D- +++ E271FG020R-S024R-N062E-S078G-G118S-N248D +++ A016S-S103A-S128N +++S024R-S078G-G118D-P129E-Q245R +++ S078D-G118D-P129E-S188D-H249R +++T022A-S024R-T033S-N062E-N116L-A158E +++A016S-T022A-S101G-S103A-V104I-N116A-S128N-Y209V-T213A- +++ A232VS024R-S078G-G118S-G159D-S188D-Q245R +++T033S-N062E-N116L-G118R-A158E-S188D +++G020R-S024R-N062E-S078D-G118S-G159D-Q245R-A272V +++G118R-S128I-A158E-S166D +++ G020K-T022L-S024F-S078N-S166D-D181E-L217E+++ N062E-G118R-S188D +++S024F-S101G-S103A-V104I-N116L-S128N-Y209A-T213A-A232V +++G020R-S078D-G118D-G159D-S188D-Q245R +++S024R-N062E-S078G-G118D-P129E-N248D-H249R +++G020R-S024R-N062E-S078G-G118D-N248D +++T022A-V068A-S101G-S103A-V104I-G159D-S188D-T213A-A232V- +++Q245R-N248D-E271F G020R-N062E-S078D-G118S-P129E-G159D-S188D-Q245R +++S024R-S078G-G118S-P129E-N248D-H249R +++T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248H- +++ E271LT033S-N116L-G118R-S188D +++ N062E-S078G-G118S-S188D-N248D-H249R +++A016S-T022A-S101G-S103A-V104I-S128N-L148I +++G020R-S078G-G118S-S188D-N248D-H249R +++G020R-S024R-S078D-G118D-P129E-G159D-Q245R-N248D +++S024F-T033S-S101G-S103A-V104I-N116L-Y209A-T213A-A232V +++S024R-N062E-G118R-S128I-A158E +++ G020R-S024R-S078D-G118S-G159D-S188D+++ S024R-N062E-S078G-G118D-G159D-Q245R-N248D-H249R +++N062E-G118R-A158E-S166D +++ G020R-S024R-S078D-G118S-P129E-S188D +++S078G-G118D-G159D +++G020R-S024R-S078D-G118D-P129E-G159D-S188D-Q245R-N248D +++G020R-S078G-G118D-S188D-N248D +++G020K-T022L-S078G-G118D-G159D-N248D-H249R +++ S103G-L148I-M175T +++G020R-S024R-S078G-G118S-P129E-Q245R-N248D +++T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245R-N248D- +++ H249R-E271FT022A-S024F-S101G-S103A-V104I-N116A-S128N-Y209A-G211Q- +++ T213A-A232VA016S-T033S-S101G-S103A-V104I-N116A-Y209A-G211Q-A232V +++T022A-S024R-N062E-S166D +++G020R-S078D-G118S-G159D-S188D-Q245R-N248D-H249R +++T022A-S024F-S101G-S103A-V104I-N116A-S128N-Y209V-A232V +++T022A-S024R-N062E-N116L-A158E-S188D +++T022Q-S101G-S103A-V104I-G159D-S188E-A232S-Q245T-N248E- +++ E271LS024R-T033S-N116L-G118R-A158E-S188D +++T022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245V-N248H- +++ E271HG020R-N062E-S078D-G118S-G159D-N248D +++ T022A-S024R-N116L-G118R-A158E+++ G020K-T022L-S024F-G118R-L217E +++S024R-S078D-G118S-G159D-S188D-N248D-H249R +++G020R-S024R-N062E-S078G-G118D-P129E-S188D-N248D +++T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D- +++ E271LG020R-S024R-N062E-S078D-G118S-P129E-G159D-S188D-N248D- +++ H249RN062E-G118R-S166D +++T022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245R-N248D +++G020R-N062E-S078D-G118S-P129E-G159D-N248D-H249R +++T022A-S101G-S103A-V104I-G159D-S188D-A232T-Q245V-N248H- +++ E271TT022L-S024F-G118R-S166D +++T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D +++G020K-S024F-S166D-T213A-L217E +++ N062E-S078G-G118S-G159D-S188D +++S024R-N062E-S078D-G118D-Q245R-N248D +++G020K-T022L-S024F-S078N-T213A-L217E +++T022A-V068A-S101G-S103A-V104I-N116L-G159D-S188D-T213A- +++A232V-Q245R-N248D-E271F N062E-S078G-G118S-P129E-G159D +++G020R-S024R-N062E-S078G-G118S-G159D-H249R +++G020K-T022L-S024F-S078N-G118R-S166D-T213A-L217E +++T022A-N062E-N116L-A158E +++T022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245V-N248E- +++ E271LT022A-S024R-N062E-S128I-S166D +++S024R-S078G-G118D-G159D-Q245R-N248D-H249R +++G020R-S024R-N062E-S078G-G118S-P129E-G159D-H249R +++G020R-S078G-G118D-S188D-Q245R-N248D +++G020R-S024R-N062E-S078G-G118D-P129E-S188D-Q245R-N248D- +++ H249RS024R-S078G-G118S-G159D-Q245R +++ T022A-S024R-N062E-G118R-S128I-A158E+++ G020R-S078D-G118S-P129E-G159D-S188D-Q245R +++S024R-S078G-G118S-Q245R-N248D-H249R +++ G118R-A158E-S188D +++G020K-S024F-G118R-S166D-L217E +++ G020K-S024F-S078N-G118R-T213A-L217E+++ T022A-S101G-S103A-V104I-G159D-S188E-A232S-Q245V-N248D- +++ E271LT022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245V-N248D +++S024R-N062E-S078G-G118S-H249R +++G020R-S024R-S078G-G118S-G159D-S188D-Q245R-N248D-H249R +++A016S-S024F-S101G-S103A-V104I-N116L-S128N-Y209V-A232V +++T033S-A158E-S166D +++ S024R-S078D-G118S-S188D-N248D-H249R +++S024R-N062E-S078D-G118S-S188D-Q245R-N248D +++N062E-S078G-G118D-G159D-Q245R-N248D +++N062E-S078G-G118D-S188D-Q245R-N248D-H249R +++S024R-N116L-G118R-S128I-A158E-S166D +++G020R-N062E-S078D-G118S-P129E-G159D-Q245R-N248D +++S024R-N062E-S101A-S103A-V104I-S188D-P210I-A232V-Q245R +++N062E-S078D-G118S-P129E-H249R +++ S024R-S078G-G118S-P129E-G159D-S188D+++ A016S-S101G-S103A-V104I-N116A-S128N-Y209V-T213A-A232V +++G020K-T022L-S024F-S078N-S166D-T213A-L217E +++G020R-S024R-N062E-S078D-G118D-S188D-N248D +++G020R-S024R-N062E-S078G-G118S-G159D-A272D +++ T022A-S024R-A158E +++G020R-S078G-G118S-G159D-N248D +++T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245V-N248E- +++ E271TS101G-S103A-V104I-N116A-Y209A-G211Q-T213A-A232V +++G020K-T022L-S024F-S166D-L217E +++ G020R-S024R-S078D-G118S-Q245R +++G020R-S078D-G118D-P129E-S188D-Q245R +++G020R-S024R-N062E-S078D-G118S-S188D-Q245R-N248D +++G020R-S024R-S078D-G118D-G159D-N248D +++T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245T-N248H +++G020R-S024R-S078D-G118S-S188D-Q245R +++G020R-N062E-S078D-G118D-S188D-Q245R +++G020R-S024R-S078D-G118D-G159D-S188D-Q245R-H249R +++T022A-S128I-A158E-S188D +++A016S-S024F-S101G-S103A-V104I-N116A-S128N-Y209V-T213A- +++ A232V-V244GT022L-S078N-T213A-L217E +++ S078D-G118S-P129E-H249R +++G020K-T022L-S024F-S078N-G118R-T213A-L217E +++N062E-S078D-G118S-G159D-Q245R +++ G020K-S024F-S166D +++S024R-N062E-S078G-G118D-Q245R-N248D-H249R +++G020K-S024F-S078N-G118R-L2I7E +++ T022L-S024F-S078N-G118R-S166D-L217E+++ T022A-S024R-T033S-A158E-S188D +++T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245T-N248D- +++ E271H S087R+++ T022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245V-N248H +++G020R-S078G-G118D-P129E-G159D-Q245R-N248D-H249R +++S078G-G118S-P129E-G159D-Q245R-N248D +++S024R-S078D-G118D-P129E-G159D-Q245R-N248D-H249R +++G020R-S078G-G118S-P129E +++ S024R-N116L-G118R-S166D-S188D +++G020R-S024R-S078G-G118S-P129E-G159D-Q245R +++ T022A-S101G-S103G-V104I+++ G020R-S024R-N062E-S078D-G118D-P129E-G159D +++G020R-S024R-N062E-S078D-G118D-P129E-Q245R-N248D-H249R +++T022A-S024R-N062E-N116L-G118R-S188D +++T022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245T-N248E- +++ E271LA016S-S101G-S103A-V104I-N116L-M119V-Y209V-G211Q-A232V +++G020R-S024R-N062E-S078G-G118D +++T022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245T-N248H- +++ E271LT022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245T-N248D- +++ E271TT033S-S101G-S103A-V104I-N116L-Y209A-G211Q-A232V +++G020R-S024R-N062E-S078D-G118S-P129E-G159D-N248D-H249R +++G020R-S078G-G118S-P129E-N248D-H249R +++ G020K-T022L-G118R-S128D +++T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245V-N248D- +++ E271LG020R-S024R-S078D-G118S-G159D-S188D-N248D-H249R +++S101G-S103A-V104I-N116L-Y209A-T213A-A232V +++T022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245T-N248E- +++ E271TS024R-N062E-S078D-G118D-G159D-Q245R-N248D-H249R +++G020K-S024F-T213A-L217E +++ G118R-S128I-S188D +++G020R-S078G-G118S-P129E-H249R +++ T022A-N062E-G118R-S188D +++S024R-S078G-G118S-P129E-G159D-Q245R +++T022A-S024F-S101G-S103A-V104I-N116L-Y209A-G211Q-T213A- +++ A232VT022A-S024R-N116L-A158E-S166D +++T022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245T-N248H- +++ E271TT022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245T-N248H- +++ E271LS024R-N062E-A158E-S188D +++ G020K-S024F-G118R-S166D-T213A-L217E +++T022A-S101G-S103A-V104I-G159D-S188E-A232S-Q245T-N248H- +++ E271TS078D-G118D-P129E-G159D-Q245R +++ S101A-S103A-V104I-S188D-A232V-Q245R+++ N062E-N116L-G118R-A158E +++T022A-T033S-S101G-S103A-V104I-N116L-Y209A-T213A-A232V +++T022L-S024F-G118R-S128D-T213A +++A016S-S024F-S101G-S103A-V104I-N116L-S128N-Y209A-A232V +++S078D-G118S-G159D-S188D-N248D-H249R +++ G020R-S078G-G118S-P129E-S188D+++ T022A-T033S-N116L-S166D +++ S024R-N062E-G118R-S188D +++S024R-T033S-N116L-G118R-S128I-A158E +++S101A-S103A-V104I-P210I-A232V-Q245R +++G020R-S024R-N062E-S078G-G118S-S188D-H249R +++T022A-S024R-N116L-G118R-S128I-A158E-S166D-S188D +++A016S-S024F-S101G-S103A-V104I-N116L-Y209V-G211Q-T213A- +++ A232VT022A-S101G-S103A-V104I-G159D-S188E-A232S-Q245V-N248D- +++ E271HS078G-G118S-G159D-S188D-N248D-H249R +++S024R-S078D-G118D-P129E-G159D-S188D-H249R +++T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245T-N248E- +++ E271FG020R-N062E-S078D-G118D +++ S024R-T033S-N116L-G118R-S128I-A158E-S188D+++ T033S-N062E-N116L-G118R-S188D +++T022A-T033S-N116L-G118R-A158E-S188D +++ S078N-G118R-S166D +++T022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245T-N248E- +++ E271LT022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245T-N248H- +++ E271LS024F-S101G-S103A-V104I-N116A-Y209A-G211Q-T213A-A232V +++G020R-S024R-S078D-G118D-G159D-S188D-Q245R-N248D +++T022A-S101G-S103A-V104I-N116L-G159D-S188D-T213A-A232V- +++Q245R-N248D-E271F T022A-G118R-A158E-S188D +++S024R-S078D-G118S-G159D-H249R +++S101G-S103A-V104I-N116L-Y209A-G211Q-A232V +++T022A-S101G-S103A-V104I-G159D-S188E-A232S-Q245V-N248E- +++ E271FS024R-N062E-S078D-G118S-P129E-Q245R +++ G020K-S024F-S078N-T213A-L217E+++ G020R-S024R-S078G-G118D-P129E-G159D-H249R +++G020R-S024R-S078D-G118S-G159D-S188D-N248D +++T022A-S101G-S103A-V104I-G118R-G159D-S188D-A232V-Q245R- +++ N248D-E271FG020R-S078G-G118S-S188D-Q245R-N248D +++G020R-S024R-N062E-S078G-G118D-P129E-S188D-N248D-H249R +++G020R-S078D-G118S-P129E-G159D-S188D-Q245R-N248D +++T022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245V-N248D- +++ E271TT022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245T-N248H +++S024R-S078D-G118S-P129E-G159D-S188D-Q245R-N248D +++ S101G-S103A-L148I+++ T022A-S101G-S103A-V104I-G159D-S188E-A232S-Q245T-N248D- +++ E271LT022A-S101G-S103A-V104I-G159D-S188D-A232T-Q245T-N248D- +++ S265G-E271LV104I-L111V-L148I +++ T022A-G118R-S128I +++G020R-S024R-S078G-G118D-P129E-G159D-N248D +++G020K-T022L-G118R-S166D-T213A-L217E +++G020R-S078D-G118D-P129E-Q245R-N248D +++ G097P-S099A-S101A-S103A +++N062E-S078G-G118S-P129E-G159D-Q245R-N248D-H249R +++ A016S-V104L-L111V+++ T022Q-S101G-S103A-V104I-G159D-S188E-A232T-Q245V-N248D- +++ E271TN062E-S078G-G118D-P129E-Q245R-N248D +++T022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245V-N248E +++T022A-S024R-T033S-G118R-A158E-S188D +++ S078N-G118R-T213A-L217E +++T022A-S024F-S101G-S103A-V104I-N116L-Y209A-G211Q-A232V +++G020R-S024R-S078G-G118D-S188D-Q245R-H249R +++T022A-S101G-S103A-V104I-N116L-S128L-G159D-S188D-A232V- +++Q245R-N248D-E271F G020K-T022L-S078N-G118R-S128D-L217E +++T022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245R-N248H- +++ E271TT022A-S024R-T033S-N116L-A158E-S188D +++ S024R-N062E-S128I-S188D +++S024R-S078G-G118S-S188D +++T022Q-S101G-S103A-V104I-G159D-S188E-A232V-Q245T-N248D- +++ E271LG020K-S024F-G118R-T213A-L217E +++T022A-S024R-S101G-S103A-V104I-P129E-G159D-S188D-A232V- +++Q245R-N248D-A270V-E271F-A272V S103A-V104I-L111V-L148I +++T022A-S024F-T033S-S101G-S103A-V104I-N116L-Y209V-G211Q- +++ T213A-A232VT022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245V-N248H- +++ E271TS078D-G118D-G159D-Q245R +++G020K-T022L-S078D-G118D-P129E-G159D-Q245R-H249R +++A016S-T022A-T033S-S101G-S103A-V104I-N116A-Y209V-G211Q- +++ T213A-A232VT022A-S024R-T033S-S166D +++ S024R-S078D-G118D-G159D-S188D-N248D-H249R+++ A016S-S024F-S101G-S103A-V104I-N116L-Y209A-T213A-A232V +++S024R-T033S-A158E +++G020R-S024R-N062E-S078G-G118D-G159D-S188D-Q245R-N248D- +++ H249RT022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245T-N248D- +++ E271TG020R-N062E-S078D-G118D-S188D-Q245R-N248D +++ S078G-G118D-S188D-Q245R+++ T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245T-N248D- +++ E271LG020R-S024R-N062E-S078G-G118D-P129E-G159D-S188D-N248D +++S024R-N116L-S128I-S166D +++ S024R-S078D-G118D-G159D-Q245R-N248D +++A016S-T022A-S024F-S101G-S103A-V104I-N116L-S128N-Y209A- +++G211Q-T213A-A232V S078G-G118D-P129E-G159D-Q245V-N248E-E271F +++G020K-S024F-S078N-G118R-S128D-T213A-L217E +++ T022L-S024F-T213A-L217E+++ T022A-S024F-S101G-S103A-V104I-N116L-G118V-Y209A-A232V +++N062E-S101A-S103A-V104I-S188D-A232V-Q245R +++ T022A-N062E-A158E +++S024R-T033S-S128I-A158E-S188D +++ S024R-S078D-G118S-S188D-Q245R +++T022Q-S101G-S103A-V104I-G159D-S188E-A232L-Q245R-N248D +++G020R-S024R-S078G-G118D-G159D-S188D-H249R +++S024R-S078G-G118D-P129E-G159D-Q245R-H249R +++S024R-N062E-S078G-G118D-P129E-G159D-S188D-N248D-H249R +++S024R-T033S-A158E-S188D +++ T022A-S101G-S103A-V104I-L148I +++N116L-G118R-S128I-S166D +++ S024R-N062E-S078D-G118S-S188D-N248D +++S078G-G118S-P129E-H249R +++ S101A-S103A-V104I-S188D-P210I-Q245R +++T022A-S101G-S103A-V104I-N116L-G118V-Y209V-T213A-A232V +++G020K-T022L-S024F-S078N-G118R-S166D +++ T022A-G118R-S128I-S188D +++G020R-N062E-S078G-G118S-N248D +++ G020R-S078D-G118D-S188D-H249R +++N062E-S078G-G118D +++ G020K-T022L-G118R-S166D-T213A +++G061E-S101G-S103A-V104I-N116A-Y209V-G211Q-T213A-A232V +++T022A-S101G-S103A-V104I-G159D-S188E-A232S-Q245V-N248E- +++ E271LT022A-S024F-T033S-S101G-S103A-V104I-N116A-Y209V-T213A- +++ A232VT022A-S024R-N076D-S101G-S103A-V104I-G159D-S188D-A232V- +++Q245R-N248D-N269D-A270V-E271F-A272VT022A-V068A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R- +++ N248D-E271FT022A-S101G-S103A-V104I-G159D-S188E-A232L-Q245T-N248E- +++ E271TS078G-G118S-P129E-S188D-N248D-H249R +++ G020K-T022L-S166D-L217E +++T213A-L217E +++ T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245V-N248D-+++ E271T T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245R-N248D +++T022A-S101G-S103A-V104I-G159D-S188E-A232L-Q245V-N248H +++G020R-S078D-G118S-G159D-Q245R-N248D-H249R +++S024F-S101G-S103A-V104I-N116A-Y209V-A232V +++T022L-S024F-G118R-S166D-L217E +++ S024R-T033S-S188D +++G020R-N062E-S078G-G118D-G159D-S188D-H249R-A272D +++ T033S-A158E-S188D+++ S101G-S103A-V104I-N116L-S128N-Y209A-G211Q-A232V +++T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245T-N248E +++G020R-S078D-G118D-G159D-Q245R +++A016S-S024F-T033S-S101G-S103A-V104I-N116L-Y209V-G211Q- +++ A232VN062E-S078D-G118S-S188D +++ G020R-S024R-S078D-G118S-P129E-S188D-Q245R+++ A016S-S101G-S128N-L148I +++ G118R-S128I-S166D-S188D +++N062E-S078D-G118D-P129E-G159D-H249R +++ T022A-T033S-N062E-N116L +++N062E-S078G-G118D-P129E-Q245R +++T022A-S024F-T033S-S101G-S103A-V104I-N116L-Y209V-T213A- +++ A232VT022A-S101G-S103A-V104I-G159D-S188D-A232T-Q245T-N248E- +++ E271LS078G-G118S-S188D-H249R +++ S024R-N062E-S078D-G118S-H249R +++S078G-G118S-P129E-S188D-N248D +++S101G-S103A-V104I-N116L-S128N-Y209V-T213A-A232V +++T022A-S024R-N062E-N116L-G118R-A158E-S188D +++G020K-S024F-S078N-S166D-L217E +++ S024R-S078G-G118S-P129E-H249R +++T022A-S024R-S128I-A158E-S166D +++S101G-S103A-V104I-N116L-Y209V-G211Q-T213A-A232V +++T022A-S024F-T033S-S101G-S103A-V104I-N116L-Y209A-G211Q- +++ A232VG020R-S078D-G118D-P129E-G159D-S188D-Q245R +++N062E-S078G-G118S-P129E-G159D-N248D-H249R +++ A016S-V104I-S128N-L148I+++ T022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245V-N248E +++T022A-G023A-T033S-S101G-S103A-V104I-N116A-Y209A-G211Q- +++ T213A-A232VT022A-N116L-S188D +++ G020R-N062E-S078D-G118D-P129E-G159D-S188D-Q245R+++ S024R-N062E-S078D-G118D-P129E-H249R +++S024F-T033S-S101G-S103A-V104I-N116L-Y209V-T213A-A232V +++S024R-S078G-G118S-G159D-S188D-N248D +++T022A-S024F-S101G-S103A-V104I-N116L-Y209V-A232V +++T022A-S101G-S103A-V104I-N116L-S128N-Y209V-G211Q-A232V +++S024R-N062E-S166D-S188D +++ S024R-S078D-G118S-G159D-S188D-Q245R-N248D+++ S101G-S103A-V104I-N116A-Y209V-T213A-A232V +++T022A-S024R-G118R-A158E-S166D-S188D +++T022A-T033S-S101G-S103A-V104I-N116L-Y209A-A232V +++G020K-T022L-S024F-S078N-G118R-S128D +++A016S-T022A-S024F-T033S-S101G-S103A-V104I-N116A-Y209V- +++ T213A-A232VS024R-N062E-N116L-S128I +++T022A-S024F-S101G-S103A-V104I-N116L-Y209A-T213A-A232V +++T022A-V068A-S101G-S103A-V104I-N116L-G159D-S188D-A232V- +++Q245R-N248D-E271F T022A-S024R-T033S-N062E-G118R-A158E +++S024R-N062E-S078D-G118D-P129E-Q245R-N248D-H249R +++T022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245R-N248D +++T022A-S024R-A158E-S188D +++ G020K-T022L-S078N-G118R-S166D-L217E +++G020R-S078D-G118D-P129E +++A016S-S101G-S103A-V104I-N116A-S128N-Y209V-G211Q-A232V +++G020K-T022L-S024F-G118R-S166D-T213A-L217E +++ G118R-T213A-L217E +++T022L-S024F-S166D-T213A-L217E +++S024R-N076D-S101A-S103A-V104I-S188D-A232V-Q245R +++T022A-T033S-G118R-P129E +++ S024R-S078G-G118S-P129E-G159D-S188D-N248D+++ T022A-S024R-S101G-S103A-V104I-I107V-P129E-G159D-S188D- +++A232V-Q245R-N248D-A270V-E271FT022A-S101G-S103A-V104I-N116L-Y209V-G211Q-T213A-A232V +++T022A-S101A-S103G-V104I-S128N +++ T022A-S024R-N062E-N116L-G118R-S166D+++ A016S-T022A-S024F-S101G-S103A-V104I-N116L-Y209A-T213A- +++ A232VA016S-S101G-S103A-V104I-N116L-Y209V-G211Q-T213A-A232V +++S024F-G118R-S128D-T213A-L217E +++ T022L-S024F-G118R-T213A-L217E +++N062E-S078D-G118S +++ S024R-G118R-A158E-S188D +++S024F-S101G-S103A-V104I-N116L-S128N-Y209A-A232V +++ A088L +++ E271W +++N062E-S078D-G118D-P129E-H249R +++ T022A-V104I-S128N-L148I +++T022Q-S101G-S103A-V104I-G159D-S188E-A230V-A232V-Q245R- +++ N248HS101G-S103A-V104I-N116L-G118V-Y209A-T213A-A232V +++S024R-S078G-G118D-G159D-Q245R-H249R +++ G097P-A098F-S099A-V104I +++T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D- +++ E271FT022L-S024F-S078N-G118R-S166D-T213A +++N062E-S078G-G118D-S188D-Q245R-N248D +++ T022A-S024R-T033S-S166D-S188D+++ T022A-T033S-R045C-G118R-A158E-S188D +++T022A-S024R-N062E-S128I-S188D +++T022A-S101G-S103A-V104I-P129E-G159D-S188D-A232V-Q245R- +++N248D-A270V-E271F T022A-S024R-N116L-S166D +++ S024R-N062E-S128I-A158E+++ S024R-G118R-A158E-S166D +++ G020K-T022L-S078N-T213A +++G020R-S024R-S078G-G118D-G159D-Q245R-N248D +++G020K-T022L-S078N-T213A-L217E +++ G020R-S078D-G118D-G159D-Q245R-H249R+++ T022A-S024F-S101G-S103A-V104I-N116L-S128N-Y209A-G211Q- +++T213A-A232V S024R-S101A-S103A-V104I-S188D-A232V-Q245R +++S024R-N116L-G118R-A158E-S166D-S188D +++ S024F-S166D-T213A +++G020R-S024R-N062E-S078D-G118S-P129E-Q245R-N248D-H249R +++T022A-S024R-S128I-S166D-S188D +++G020R-S024R-S078D-G118S-P129E-N248D-H249R +++T022A-S024R-T033S-N116L-G118R-A158E-S166D-S188D +++T022A-S024F-T033S-S101G-S103A-V104I-N116L-S128N-Y209A- +++ T213A-A232VT022L-S024F-L217E +++ G020R-S078G-G118S-G159D-S188D-Q245R-N248D-H249R+++ T022A-S101G-S103A-V104I-N116L-G159D-S188D-A232V-Q245R- +++N248D-E271F S099T-S103G +++ S024R-S078D-G118D-P129E-N248D-H249R +++S024R-S078D-G118D-P129E-S188D-Q245R-N248D +++T022A-T033S-S101G-S103A-V104I-G118R-G159D-S188D-T213A- +++A232V-Q245R-N248D-E271F S024R-S078D-G118S-P129E-G159D-S188D-N248D-H249R+++ S078D-G118D-P129E-G159D-Q245R-H249R +++A016S-T022A-S024F-S101G-S103A-V104I-N116A-Y209A-A232V +++S024R-S078D-G118D-P129E-G159D-S188D-Q245R-N248D-H249R +++T022A-S101G-S103A-V104I-G118R-S128L-G159D-S188D-T213A- +++A232V-Q245R-N248D-E271F G020R-S078G-G118S-G159D-Q245R-N248D +++S078G-G118D-G159D-S188D-Q245R +++ T022L-G118R-T213A-L217E +++G020R-S024R-S078G-G118S-S188D-N248D-H249R +++ G118R-A158E-S166D-S188D+++ A016S-T022A-S024F-T033S-S101G-S103A-V104I-N116L-Y209V- +++T213A-A232V N062E-S078D-G118S-P129E-G159D-Q245R-N248D +++T022Q-A088S-S101G-S103A-V104I-G159D-S188D-A232T-Q245V- +++ N248ES078D-G118S-P129E-G159D-Q245R-N248D +++ G020K-S024F-S128D-N204K +++S024F-S101G-S103A-V104I-N116A-Y209V-G211Q-A232V +++S024R-S078D-G118S-S188D +++ S101G-S103A-V104I-N116L-Y209V-A232V +++G020R-N062E-S078D-G118S-S188D-Q245R-N248D-H249R +++A016S-T022A-S024F-S101G-S103A-V104I-N116L-Y209A-G211Q- +++ A232VS101G-S103A-V104L-L148I +++T022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245T-N248D- +++ E271LS024R-N062E-S078D-G118D-P129E-S188D-H249R +++ R010H-T022L-S024F-L217E+++ T022A-S101G-S103A-V104I-G159D-S188E-A232L-Q245R-N248D +++S024R-N062E-S078G-G118D-S188D-Q245R +++ S024R-N116L-S128I-A158E-S166D+++ T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245T-N248H- +++ E271FG020R-S078D-G118S-P129E-G159D +++G020R-S024R-S078D-G118D-P129E-Q245R-N248D +++S024R-N076D-S101A-S103A-V104I-P210I-A232V-Q245R +++S078D-G118D-S188D-Q245R +++ G020K-T022L-S024F-G118R-S166D-T213A +++S078G-G118S-G159D-Q245R-N248D-H249R +++T022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245R-N248E- +++ E271FS056R-G065P +++ S056K +++T022A-S024R-N076D-S101G-S103A-V104I-P129E-G159D-S188D- +++A232V-Q245R-N248D-E271F T022A-S024R-N062E-N116L-G118R-S128I-A158E +++S024R-S078G-G118S-P129E-S188D +++G020R-N062E-S078D-G118D-G159D-N248D-H249R +++S024R-S078G-G118S-G159D-N248D +++ S024R-N062E-S078G-G118D-S188D-N248D+++ G020R-S024R-N062E-S078D-G118S-P129E-Q245R +++S024R-S078D-G118D-G159D +++ T022A-S024R-T033S-G118R-S128I-S166D-S188D+++ A016S-T033S-S101G-S103A-V104I-N116L-S128N-Y209V-A232V +++T022A-T033S-S188D +++ G097P-S103A-V104L +++ G020R-S078G-G118D-P129E +++N062E-S101A-S103A-V104I-S188D-V203L-A232V-Q245R +++G020R-S024R-S078G-G118S-G159D-Q245R-N248D +++G020R-S078D-G118D-G159D-S188D +++G020R-S024R-S078G-G118D-S188D-Q245R-N248D +++G020R-N062E-S078D-G118D-S188D +++ G020R-S101A-P210I-G211Q +++T022A-T033S-N116L-S188D +++T022A-T033S-S101G-S103A-V104I-G159D-S188D-A232V-Q245R- +++ N248D-E271FT022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245R-N248E- +++ E271LS024R-N062E-S078D-G118D-N248D-H249R +++ T022A-S024R-N116L-A158E +++N062E-N076D-S101A-S103A-V104I-S188D-P210I-A232V-Q245R +++S024R-S078G-G118S-S188D-N248D-H249R +++G020R-S078G-G118S-P129E-S188D-N248D-H249R +++S024R-S078D-G118D-G159D-S188D +++A016S-S101G-S103A-V104I-N116L-Y209A-G211Q-A232V +++ S132D +++ N043K +++G097P-V104L +++ A016S-T022A-S024F-S101G-S103A-V104I-N116A-Y209A-G211Q-+++ A232V S024R-N062E-S078D-G118D-G159D-S188D-H249R +++S024R-S078G-G118S-G159D-S188D +++ S024R-S078G-G118D-G159D-S188D-N248D+++ S024R-N062E-S078G-G118S-G159D-S188D-Q245R-H249R +++S024F-S101G-S103A-V104I-N116A-Y209V-T213A-A232V +++G020K-S078N-S166D-T213A-L217E +++ S103A-L111V-L148I +++S024F-T033S-S101G-S103A-V104I-N116L-S128N-Y209A-G211Q- +++ T213A-A232VS024R-S078D-G118D-G159D-S188D-Q245R-N248D +++T022A-T033S-S101G-S103A-V104I-N116A-Y209V-T213A-A232V +++S024R-N062E-S078G-G118D-P129E-S188D +++S024R-S078D-G118D-P129E-S188D-Q245R-H249R +++ T022A-G118R-S166D +++G020R-S024R-S078G-G118S-G159D-S188D-Q245R +++T022A-S024R-T033S-N116L-G118R-A158E-S188D +++T022Q-S101G-S103A-V104I-G159D-S188E-A232T-Q245V-N248D- +++ E271FG020R-S024R-S078D-G118S-P129E-Q245R-N248D-H249R +++A016S-T022A-V104I-S128N +++S024R-N062E-N076D-S101A-S103A-V104I-S188D-P210I-A232V- +++ Q245RT022L-G118R-S166D-T213A-L217E +++S078D-G118S-P129E-G159D-S188D-Q245R-H249R +++ G195W-N269E +++G020R-S024R-S078G-G118S-P129E-Q245V-N248E +++ N269W +++T022A-T033S-G118R-A158E-S166D-S188D +++G020R-N062E-S078G-G118D-P129E-G159D-Q245R-H249R +++ A016S-S101A-S103A+++ G097P-S099G-S103A-V104I +++ S024R-S078D-G118D-P129E-Q245R +++T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245R-N248E +++S078G-G118D-P129E-S188D-Q245R +++ T033S-N062E-G118R-A158E +++G020R-S078D-G118S-G159D-N248D +++ A016S-T022A-S103G-V104I-S128N-L148I+++ A016S-S101G-S103A-V104I-N116A-Y209A-A232V +++S101A-S103A-V104I-S188D-M222S-A232V-Q245R +++T022A-S024F-S101G-S103A-V104I-N116A-S128N-Y209V-T213A- +++ A232VA016S-T022A-S024F-S101G-S103A-V104I-N116A-Y209A-T213A- +++ A232VT022A-S101G-S103A-V104I-G159D-S188D-T213A-A232V-Q245R- +++ N248D-E271FA016S-S101G-S103A-V104I-N116L-Y209A-T213A-A232V +++G020R-S078D-G118S-S188D +++ G020R-S078D-G118S-P129E-Q245R-N248D +++T022L-S166D-T213A +++ G020R-S024R-S078D-G118S-P129E-G159D-S188D-N248D+++ S240G +++ L217E +++ S078G-G118S-P129E-Q245R-N248D +++S024R-S078D-G118D-P129E-G159D-Q245R-N248D +++ S024R-S078G-G118S-G159D+++ T022A-S024R-N076D-S101G-S103A-V104I-G159D-S188D-A232V- +++Q245R-N248D-E271F-A272V N062E-S078G-G118S-P129E-S188D-Q245R-N248D-H249R+++ S024F-S078N-G118R-S166D +++ S078G-G118S-P129E-N248D +++ N155W +++A016S-S024F-S101G-S103A-V104I-N116A-S128N-Y209V-G211Q- +++ T213A-A232VG020R-N062E-S078D-G118D-P129E-Q245R-N248D +++G020R-S078D-G118D-P129E-S188D-Q236R-N248D-H249R +++T022A-S024R-N116L-A158E-S188D +++S101G-S103A-V104I-N116A-S128N-Y209V-G211Q-T213A-A232V +++S078D-G118S-S188D-Q245R-N248D +++ T022L-S024F-S078N-L217E +++S099A-S101A-G102A +++ G020R-S078D-G118D-G159D-N248D-H249R +++T022A-N076D-S101G-S103A-V104I-I107V-P129E-G159D-S188D- +++A232V-Q245R-N248D-E271F T033S-N116L-G118R-S166D-S188D +++S024R-G118R-S128I-A158E-S166D-S188D +++G020R-S024R-N062E-S078G-G118S-P129E-G159D-Q245R-N248D- +++ H249RN062E-S078G-G118D-N248D-H249R +++ S024R-N116L-A158E-S188D ++G020K-S024F-S078N-G118R-T213A ++ T022A-S024R-T033S-N116L-G118R-S128I ++G020R-S078D-G118D-S188D-Q245R ++S024F-S101G-S103A-V104I-N116A-Y209A-T213A-A232V ++S024R-N062E-S078G-G118D-G159D-S188D ++T022A-N062E-N116L-G118R-A158E-S166D ++ G020K-T022L-S078N-S166D-L217E ++G020R-S078D-G118D-G159D ++ T022A-S024R-T033S-N116L ++G020R-S078D-G118D-P129E-G159D ++ S166D ++ G020R-S078D-G118D-G159D-H249R++ S024R-N062E-S078G-G118S ++ S024R-S078D-G118D-S188D-Q245R-H249R ++A016S-S024F-T033S-S101G-S103A-V104I-N116L-Y209V-T213A- ++ A232VG020R-N062E-S078D-G118D-P129E-G159D-Q245R ++S024F-S101G-S103A-V104I-N116L-G118V-Y209V-G211Q-A232V ++S024F-S166D-L217E ++ T022A-S024R-S166D ++S024R-N062E-S078G-G118D-P129E-N248D ++A016S-S101G-S103A-V104I-N116L-S128N-Y209V-A232V ++A016S-T022A-S101G-S103A-V104I-N116A-Y209V-G211Q-A232V ++S078D-G118S-G159D-N248D-H249R ++ S024R-S078G-G118D-P129E-G159D-S188D ++T022A-S024R-N062E-N116L-G118R-S128I-S188D ++ S103A-S128N ++T022L-S078N-G118R-S166D-T213A ++S024F-T033S-S101G-S103A-V104I-N116L-S128N-Y209A-T213A- ++ A232VT022A-S024R-N076D-S101G-S103A-V104I-P129E-G159D-S188D- ++A232V-Q245R-N248D-E271F-A272V S024R-S078D-G118S-S128N-P129E ++S024R-S078G-G118S-P129E-G159D-S188D-Q245R-H249R ++T022A-S024R-N076D-S101G-S103A-V104I-P129E-G159D-S188D- ++A232V-Q245R-N248D-A270V-E271F-A272VT022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245T-N248D ++S024R-T033S-N116L-A158E ++ S130E-N269K ++ S024R-T033S-N116L-S128I-A158E++ A016V-G020R-S024R-S078D-G118D-P129E-Q245R-N248D ++S099G-S101A-G102A-S103G-V104I ++ A016S-T022A-S101A-V104L-S128N-L148I ++G020R-S024R-S078G-G118S-P129E-S188D ++ S259W ++ S259H ++T022A-S101G-S103A-V104I-P129E-G159D-S188D-A232V-Q245R- ++ N248D-E271FN018D-S101G-S103A-V104I-N116A-Y209A-G211Q-T213A-A232V ++G020K-S024F-S078N-S166D-T213A ++N076D-S101A-S103A-V104I-S188D-A232V-Q245R ++S078D-G118D-P129E-G159D-H249R ++ S024R-S078G-G118D-P129E-G159D-N248D ++S024R-N062E-S101A-S103A-V104I-S188D-A232V-Q245R ++ E271R ++G020R-S024R-S078D-G118D-P129E-G159D-S188D ++T022A-S024R-N076D-S101G-S103A-V104I-G159D-S188D-A232V- ++Q245R-N248D-A270V-E271F G020R-N062E-S078D-G118S-G159D-S188D-Q245R-H249R++ Q012R-S101G-S103A-V104I-N116L-S128N-Y209V-A232V ++G020R-S078G-G118D-P129E-G159D-S188D-Q245R-N248D ++G020R-N062E-S078G-G118S-P129E-G159D-S188D-Q245R-H249R ++T022A-S101G-S103A-V104I-G118R-S128L-G159D-S188D-A232V- ++Q245R-N248D-E271F G020R-S078D-G118D-G159D-S188D-Q245R-N248D ++T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D- ++ E271F-A272VG020R-S024R-N062E-S078D-G118S-P129E-S188D-Q245R-N248D- ++ H249RG020R-S078G-G118S-G159D-H249R ++ G020K-T022L-S024F-G118R-S128D-S166D ++T022A-S024R-N062E-N116L-G118R-S128I-A158E-S166D ++ A016S-S103G-V104L ++A016S-T033S-S101G-S103A-V104I-N116L-Y209A-A232V ++G020R-S024R-N062E-S078D-G118S-P129E-S188D-N248D ++G020K-T022L-S078N-G118R-S166D ++ G020K-T213A-L217E ++S024R-N062E-S078D-G118S-P129E-G159D-S188D-Q245R-N248D- ++ H249RS078N-S166D-T213A-L217E ++ S078D-G118S-P129E-G159D-Q245R-H249R ++T022A-N076D-S101G-S103A-V104I-P129E-G159D-S188D-A232V- ++Q245R-N248D-E271F-A272V G020K-T022L-G118R-L217E ++ T022A-V104L-L111V ++T022A-S101G-S103A-V104I-S128A-P129E-G159D-S188D-A232V- ++Q245R-N248D-E271F S024R-N062E-S078G-G118S-P129E-S188D-N248D ++G020R-S024R-S078D-G118D-G159D-S188D-N248D ++ T022L-S078N-L217E ++G020R-S024R-S078G-G118S-S188D ++S024F-S101G-S103A-V104I-N116L-Y209V-G211Q-T213A-A232V ++T022A-N116L-G118R-A158E-S166D ++G020R-S024R-S078G-G118S-P129E-S188D-Q245R-N248D-H249R ++T022A-V104I-S128N ++T022A-S101G-S103A-V104I-N116L-S128N-Y209V-G211Q-T213A- ++ A232VS024R-S078G-G118D-Q245R ++ N062E-S078G-G118S-P129E-G159D-S188D-H249R ++T022A-S101G-S103A-V104I-I107V-G159D-S188D-A232V-Q245R- ++N248D-A270V-E271F-A272VS024R-N062E-S078D-G118S-P129E-G159D-Q245R-N248D-H249R ++T022A-T033S-G118R-A158E-S188DT022A-S078N-G097A-S101N-S103A-V104I-S128A-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271F T022A-S024R-S166D-S188D ++A016S-S024F-T033S-S101G-S103A-V104I-N116L-Y209A-A232V ++T022A-T033S-S128I-A158E ++ S024R-T033S-N116L-S188D ++N062E-N076D-S101A-S103A-V104I-P210I-A232V-Q245R ++T022A-N076D-S101G-S103A-V104I-G159D-S188D-A232V-Q245R- ++N248D-A270V-E271F-A272V S130D ++G020K-T022L-S024F-S078N-G118R-S166D-L217E ++T022Q-S101G-S103A-V104I-G159D-S188E-A232S-Q245V-N248E ++S024R-T033S-N116L-S166D-S188D ++ S024F-S078N-T213A-L217E ++S024R-S078D-G118S-S188D-Q245R-H249R ++T022A-S024R-T033S-A158E-S166D-S188D ++ N116L ++G020K-T022L-S024F-S166D-T213A-L217E ++ T022A-N062E-N116L-G118R-S188D ++G020R-S024R-S078G-G118S-G159D ++ G020R-S078G-G118D-Q245R-N248D-H249R ++T022A-N116L-G118R-S128I ++ T022L-S078N-S166D ++ T022L-G118R-S128D ++G097P-S099A ++ T022L-S024F-G118R-S128D ++ S242K ++N062E-S078D-G118S-G159D ++ A016S-S103G-S128N-L148I ++ G097P-A098Q-S101A++ T022A-S103A-V104L-S128N-L148I ++T022A-S101G-S103A-V104I-G118R-G159D-S188D-T213A-A232V- ++Q245R-N248D-E271F S099G-S101G-G102A ++T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245T-N248D ++G100S-S101G-V104I ++ S078D-G118S-G159D-Q245R-N248D ++A016S-S024F-T033S-S101G-S103A-V104I-N116A-Y209V-G211Q- ++ T213A-A232VT022A-S024R-T033S-N116L-A158E ++T022A-N076D-S101G-S103A-V104I-G159D-S188D-A232V-Q245R- ++N248D-E271F-A272V T022A-S024R-N116L-S128I-S166D ++S101G-S103A-V104I-N116L-S128N-Y209V-A232V ++S024F-S101G-S103A-V104I-N116L-Y209A-A232V ++G020R-S078G-G118D-G159D-S188D-N248D ++G020R-S078D-G118D-S188D-Q245R-N248D ++ S024F-S078N-G118R-S128D ++T022L-S024F-S078N-G118R-S166D ++G020R-S024R-S078D-G118S-P129E-G159D-S188D-Q245R-H249R ++S103A-V104L-S128N ++ T022A-S024R-N062E-N116L-S128I ++T022A-N062E-N116L-G118R-A158E-S188D ++T022A-S024G-G097S-S101G-S103A-V104I-S128A-G159D-S188D- ++A232V-Q245R-N248D-E271FG020R-S024R-N062E-S078D-G118D-G159D-S188D-Q245R-H249R ++N062E-S078D-G118S-S188D-Q245R-N248D ++ T022A-S103A-L148I ++T022A-S024R-N116L-S128I-A158E-S188D ++ G097P-S099T-S101G-V104I ++S166D-T213A-L217E- ++ A016S-T033S-S101G-S103A-V104I-N116L-Y209V-A232V ++N062E-S078D-G118S-P129E-G159D-S188D-Q245R ++T022A-S024F-S101G-S103A-V104I-N116A-S128N-Y209A-G211Q- ++ A232VG020K-T022L-S078N-G118R-S128D-S166D-L217E ++ T033S-S188D ++T033S-N116L-G118R-A158E ++T022A-S024G-N076D-V093I-S101N-S103A-V104I-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271F G020R-S078D-G118S-P129E-Q245R-H249R ++T022A-S024R-A158E-S166D-S188D ++ G020K-T022L-S078N-G118R-S128D ++G020R-S024R-S078D-G118D-P129E-S188D ++ S099T-G102A ++S024R-N116L-G118R-A158E-S188D ++ T033S-N116L-G118R-S166D ++T022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245V-N248H ++G020R-S024R-S078D-G118S-G159D-Q245R ++ S078D-G118D-Q245R ++G020K-S078N-G118R-S166D-T213A-L217E-A272D ++A016S-T022A-V104L-L111V-L148I ++T022Q-S101G-S103A-V104I-G159D-S188D-A232V-Q245V-N248H ++N062E-N116L-G118R-S166D ++ N062E-S166D ++T022A-S024R-N062E-G118R-A158E-S188D ++T022A-S024F-S101G-S103A-V104I-N116A-Y209V-A232V ++T022A-S024R-N076D-S101G-S103A-V104I-P129E-G159D-S188D- ++A232V-Q245R-N248D-A270V-E271F G020R-S024R-N062E-S078G-G118D-Q245R ++S024R-N116L-S128I-A158E-S188D ++S024R-N062E-S101A-S103A-V104I-P210I-A232V-Q245R ++ A114T ++T022L-S024F-S078N-G118R-S128D ++ S024R-N116L-G118R-S166D ++N062E-S078G-G118D-P129E ++A016S-T022A-S101G-S103A-V104I-N116L-Y209A-G211Q-A232V ++G020K-T022L-S078N-S166D ++G020R-S024R-N062E-S078G-G118S-P129E-S188D-Q245R-N248D- ++ H249RT022A-S078N-G097A-S101Q-S103A-V104I-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271F T022A-S101G-S103G ++A016S-S024F-S101G-S103A-V104I-N116L-S128N-Y209A-G211Q- ++ S212L-A232VS024F-G118R-S166D-T213A ++ G100S-S101G ++T022A-S101G-S103A-V104I-S128L-G159D-S188D-T213A-A232V- ++Q245R-N248D-E271F A016S-T022A-S103A-V104L-S128N ++G020K-S024F-S078N-G118R-S166D ++ S099T-G102A-S103G ++G020K-S024F-S128D-L217E ++ S078D-G118D-G159D-H249R ++N062E-S078G-G118D-S188D ++ N076D-S101A-S103A-V104I-P210I-A232V-Q245R ++S024R-S078G-G118D-P129E-N248D ++ S078G-G118S-G159D-S188D ++A016S-S101G-S103A-V104I-N116L-S128N-Y209V-G211Q-T213A- ++ A232VG020K-S078N-G118R-S166D-T213A-L217E ++T022A-S101N-S103A-V104I-L124V-P129S-G159D-S188D-A232V- ++Q245R-N248D-E271F T022A-S101N-S103A-V104I-P129S-G159D-S188D-L217Q-A232V-++ Q245R-N248D-E271F S024R-S078D-G118D-P129E ++ G020K-S024F-S128D-T213A++ T022A-T033S-A158E-S188D ++ G020K-S024F-S128D-S166D-T274A ++S024R-N062E-N116L-G118R-S128I-A158E-S166D-S188D ++S078G-G118S-P129E-S188D ++ T022A-S024R-S128I-A158E-S188D ++T022A-S024R-G118R-S128I-A158E ++ T022A-N116L-S166D ++G020K-G118R-S128D-S166D ++ G020K-T022L-S024F-S078N-G118R-S128D-L217E ++G020R-S024R-S078D-G118D-P129E-S188D-N248D ++G020K-T022L-G118R-S128D-L217E ++ S024F-S078N-G118R-S128D-T213A ++S024R-S078G-G118D-S188D-N248D ++N062E-N076D-S101A-S103A-V104I-S188D-A232V-Q245R ++T033S-G118R-S128I-A158E-S166D ++A016S-S101G-S103A-V104I-N116A-S128N-Y209V-A232V ++T022L-S078N-G118R-S128D-L217E ++ S101G-V104I-L111V-S128N ++T022A-S078N-G097S-S101N-S103A-V104I-S128A-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271FT022Q-S101G-S103A-V104I-G159D-S188D-A232T-Q245V-N248E ++T022A-S078N-G097A-S101N-S103A-V104I-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271F G020K-T022L-S024F-G118R-S128D ++T022A-N076D-S078N-G097S-S101G-S103A-V104I-G159D-S188D- ++A232V-Q245R-N248D-E271FT022A-S024R-S101G-S103A-V104I-P129E-G159D-S188D-A232V- ++Q245R-N248D-A270V-E271F V104I ++T022A-S101G-S103A-V104I-P131L-G159D-S188D-A232V-Q245R- ++ N248D-E271FT022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245T-N248E ++T022A-N076D-S078N-S101N-S103A-V104I-S128A-G159D-S188D- ++A232V-Q245R-N248D-E271FA016S-S024F-T033S-S101G-S103A-V104I-N116L-Y209A-T213A- ++ A232VT022A-S101G-S103A-V104I-N116L-S128N-Y209V-T213A-A232V ++T022A-S024R-T033S-N062E-G118R-A158E-S188D ++T022A-S024R-S101G-S103A-V104I-G159D-S188D-A232V-Q245R- ++ N248D-E271FT022A-N116L-G118R-S188D ++T022A-G097A-S101Q-S103A-V104I-L124V-S128A-P129S-G159D- ++S188D-A232V-Q245R-N248D-E271FT022A-S101G-S103A-V104I-N116L-Y209V-T213A-A232V ++ T022A-S128I-S188D ++G020R-S024R-S078G-G118D-G159D ++ S024R-T033S-N116L-S128I-A158E-S188D ++T022A-T033S-S101G-S103A-V104I-G159D-S188D-T213A-A232V- ++Q245R-N248D-E271F A016S-T033S-S101G-S103A-V104I-N116L-S128N-Y209A-A232V++ A016S-S101G-S103G-V104I-L148I ++ S024R-T033S-G118R-S166D-S188D ++T022A-T033S-S101G-S103A-V104I-N116L-Y209V-T213A-A232V ++T022A-N062E-N116L-S128I ++ A016S-S101G-S103G-V104L-L148I ++T022A-N116L-S166D-S188D ++ G020K-T022L-S024F-S078N-S128D-T213A ++T022A-S101G-S103A-V104I-N116L-Y209V-G211Q-A232V ++ S099G-G100S-V104L ++S078G-G118D-P129E-G159D-S188D-H249R-T253M ++A016S-T022A-S101G-S103A-V104I-N116A-S128N-Y209A-A232V ++T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245T-N248E ++G020R-S078D-G118D-P129E-G159D-S188D-N248D-H249R ++G118R-S166D-T213A-L217E ++ G020K-S024F-S128D ++T022A-S024F-S101G-S103A-V104I-N116L-S128N-Y209V-G211Q- ++ T213A-A232VT022A-G097S-S101Q-S103A-V104I-S128A-G159D-S188D-A232V- ++Q245R-N248D-E271F G020K-T022L-G118R-S128D-T213A-L217E ++S024F-G118R-S128D-T213A ++ T022A-S101A-S103A-V104I-S128N ++S024R-N062E-N076D-S101A-S103A-V104I-S188D-A232V-Q245R ++T022A-S103A-S128N-L148I ++ G020R-N062E-S078G-G118D-G159D-S188D-N248D ++T022A-S024R-T033S-G118R-S128I-S188D ++T022A-S101G-S103A-V104I-S128L-G159D-S188D-A232V-Q245R- ++ N248D-E271FS101G-S103A-V104I-N116L-S128N-Y209V-G211Q-A232V ++T022A-S101Q-S103A-V104I-P129E-G159D-S188D-L217Q-A232V- ++Q245R-N248D-E271F T022A-S024G-S078N-G097A-S101N-S103A-V104I-S128A-G159D-++ S188D-L217Q-A232V-Q245R-N248D-E271FT022A-G097A-S101N-S103A-V104I-P129S-G159D-S188D-A232V- ++Q245R-N248D-E271F T033S-S166D-S188D ++ S078D-G118S-P129E-Q245R-N248D ++G020K-S024F-S078N-G118R-S166D-T213A ++S024R-N062E-N116L-G118R-S166D-S188D ++G020R-N062E-S078D-G118D-P129E-G159D-N248D-H249R ++G020K-S078N-G118R-S128D ++ T022A-N062E-S166D ++ P014R ++ S240D ++ N155G++ A016S-T022A-S024F-S101G-S103A-V104I-N116A-S128N-Y209A- ++ T213A-A232VA016S-T022A-T033S-S101G-S103A-V104I-N116L-Y209V-G211Q- ++ A232VA016S-T022A-S024F-T033S-S101G-S103A-V104I-N116L-Y209A- ++G211Q-T213A-A232V T022A-G097S-S101N-S103A-V104I-G159D-S188D-A232V-Q245R-++ N248D-E271F A016S-S101G-S103A-V104I-N116L-S128N-Y209V-G211Q-A232V ++G118R-S128I-S166D ++T022A-S078N-G097S-S101N-S103A-V104I-S128A-G159D-S188D- ++A232V-Q245R-N248D-E271FT022A-S101G-S103A-V104I-M119V-P129E-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271F S024R-A158E-S188D ++T022A-S101N-S103A-V104I-L124V-P129E-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271F N116L-G118R-S188D ++T022A-G097A-S101N-S103A-V104I-L124V-S128A-G159D-S188D- ++A232V-Q245R-N248D-E271F T022A-G118R-S128I-A158E ++ G097P-S101A-V104L ++S103G-V104I-S128N-L148I ++ G020R-N062E-S078G-G118D-P129E-S188D ++ V026W++ G020R-N062E-S078D-G118D-P129E-G159D ++T022A-N076D-S101G-S103A-V104I-I107V-N123S-P129E-G159D- ++S188D-A232V-Q245R-N248D-E271F-A272V S103G-S128N-L148I ++T022A-S024R-N076D-S101G-S103A-V104I-G159D-S188D-A232V- ++Q245R-N248D-E271F T022A-S024G-N076D-S078N-S101G-S103A-V104I-S128A-G159D-++ S188D-L217Q-A232V-Q245R-N248D-E271FT022A-S078N-G097S-S101G-S103A-V104I-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271FT022A-N076D-S101N-S103A-V104I-G159D-S188D-L217Q-A232V- ++Q245R-N248D-E271F N062E-S078D-G118S-G159D-S188D-Q245R-H249R ++S024R-N076D-S101A-S103A-V104I-A232V-Q245R ++ T022L-S078N ++A016S-T022A-S024F-S101G-S103A-V104I-N116L-Y209V-T213A- ++ A232VG020K-T022L-S128D-T213A ++ N062E-G118R-S128I-S188D ++S024R-N116L-S128I-S166D-S188D ++T022A-S101N-S103A-V104I-S128A-P129E-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271F S024R-T033S-N116L-G118R-A158E ++T022A-N076D-G097A-S101Q-S103A-V104I-G159D-S188D-A232V- ++Q245R-N248D-E271F T022L-S024F-S166D-L217E ++S024R-S078D-G118S-G159D-N248D-H249R ++T022A-S101Q-S103A-V104I-P129S-G159D-S188D-A232V-Q245R- ++ N248D-E271FT022A-N076D-S101G-S103A-V104I-S128A-G159D-S188D-A232V- ++Q245R-N248D-E271F A016S-T022A-S024F-S101G-S103A-V104I-N116A-S128N-Y209A-++ A232V S078G-G118D-P129E-Q245R-N248D ++ G020R-S078G-G118D-G159D-N248D++ G020K-T022L-G118R-S128D-S166D ++T022A-S024G-S101Q-S103A-V104I-S128A-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271FT022A-S024G-N076D-S078N-G097A-S101G-S103A-V104I-P129E- ++G159D-S188D-L217Q-A232V-Q245R-N248D-E271FT022A-S101G-S103A-V104I-G159D-S188E-A232T-Q245T-N248D ++G020R-N062E-S078G-G118D-S188D-Q245R-H249R ++S024F-S101G-S103A-V104I-N116L-Y209A-T213A-A232V ++S024R-N062E-N076D-S101A-S103A-V104I-A232V-Q245R ++T022A-S024R-N116L-G118R-S128I-A158E ++ A098Q-S099T-S101A-V104L ++T022A-S024G-G097S-S101G-S103A-V104I-G159D-S188D-A232V- ++Q245R-N248D-E271F T022A-S078N-S101G-S103A-V104I-S128A-G159D-S188D-L217Q-++ A232V-Q245R-N248D-E271FT022A-S101G-S103A-V104I-P129E-G159D-S188D-L217Q-A232V- ++Q245R-N248D-E271F S099G-G102A ++T022A-N076D-S101G-S103A-V104I-G159D-S188D-A232V-Q245R- ++N248D-A270V-E271F T022A-S078N-G097S-S101N-S103A-V104I-G159D-S188D-L217Q-++ A232V-Q245R-N248D-E271FT022A-N076D-S101G-S103A-V104I-G159E-S188D-A232V-Q245R- ++ N248D-E271FT022A-G097A-S101G-S103A-V104I-P129E-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271FT022A-N076D-S101N-S103A-V104I-S128A-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271F-T274A A016S-V104I-L148I ++ G097P-S101A-S103A ++G020K-T022L-S024F-G118R-S166D ++T022A-S024R-N062E-N116L-G118R-S128I-S166D ++A016S-S024F-T033S-S101G-S103A-V104I-N116A-Y209A-G211Q- ++ T213A-A232VT022A-S101Q-S103A-V104I-L124V-P129E-G159D-S188D-A232V- ++Q245R-N248D-E271F T022A-N076D-S101G-S103A-V104I-S128A-G159D-S188D-L217Q-++ A232V-Q245R-N248D-E271FT022A-S024G-N076D-S078N-S101G-S103A-V104I-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271F N062E-S078G-G118S-P129E-N248D ++G020R-S078D-G118S ++T022A-S101Q-S103A-V104I-L124V-S128A-P129E-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271F G097P-S101G-S103A-V104I ++G020K-T022L-S166D-T213A-L217E ++T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D- ++ A270V-E271FT022A-S078N-S101N-S103A-V104I-P129E-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271F A016S-S101A-S103A-S128N ++T022A-S101G-S103A-V104I-G159D-S188D-L217Q-A232V-Q245R- ++ N248D-E271FA098Q-S099T-G102A-S103A ++T022A-N076D-S078N-G097A-S101G-S103A-V104I-S128A-G159D- ++S188D-L217Q-A232V-Q245R-N248D-E271F S078N-G118R-H120N-S128D-S166D ++S101G-S103G-L148I ++ T022A-S024R-T033S-A158E-S166D ++ N116L-G118R-S166D++ T022A-N062E-N116L-S166D ++N076D-S101A-S103A-V104I-S188D-P210I-M222S-A232V-Q245R ++T022A-S101G-V104L-L148I ++T022A-S101G-S103A-V104I-G159D-S188E-Q245V-N248D ++S024R-N076D-S101A-S103A-V104I-S188D-P210I-M222S-A232V- ++ Q245RT022A-S024G-N076D-G097S-S101N-S103A-V104I-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271F N116L-G118R ++T022A-S024G-S101N-S103A-V104I-G159D-S188D-L217Q-A232V- ++Q245R-N248D-E271F T033S-S101G-S103A-V104I-N116L-S128N-Y209A-G211Q-T213A-++ A232V T022L-G118R-S166D-T213A ++A016S-T022A-S101G-S103A-V104I-N116A-Y209V-A232V ++T022A-N076D-S101G-S103A-V104I-P129E-G159D-S188D-A232V- ++Q245R-N248D-A270V-E271F T022A-S101G-S103G-V104L-L148I ++S024R-S078G-G118D-S188D ++T022A-S101G-S103A-V104I-G159D-S188D-A232V-N238Y-Q245R- ++N248D-A270V-E271F-A272VT022A-S024G-S078N-S101Q-S103A-V104I-G159D-S188D-S216G- ++L217Q-A232V-Q245R-N248D-E271F S024R-N116L-S128I ++P086T-G097P-S099T-S103A-V104I ++ A098F-G102A ++T022A-S101Q-S103A-V104I-L126I-S128A-P129E-G159D-S188D- ++A232V-Q245R-N248D-E271F N062E-S078G-G118D-G159D-N248D ++T022A-S024G-N076D-G097S-S101G-S103A-V104I-S128A-G159D- ++S188D-L217Q-A232V-Q245R-N248D-E271FA016S-T022A-S024F-S101G-S103A-V104I-N116L-S128N-Y209V- ++ G211Q-A232VG020R-S024R-S078D-G118S-H249R ++ G020K-S078N-G118R-S166D-T213A ++G020K-T022L-S024F-G118R ++ S024R-S101A-S103A-V104I-P210I-A232V-Q245R ++T022A-N076D-G097A-S101N-S103A-V104I-S128A-G159D-S188D- ++A232V-Q245R-N248D-E271F G097P-A098Q-S099G-S103A ++T022A-S024G-N076D-G097S-S101N-S103A-V104I-S128A-G159D- ++S188D-L217Q-A232V-Q245R-N248D-E271FT022A-S078N-S101N-S103A-V104I-G159D-S188D-L217Q-A232V- ++Q245R-N248D-E271F T022A-S024G-N076D-S078N-S101N-S103A-V104I-G159D-S188D-++ A232V-Q245R-N248D-E271F G020K-G118R-S128D-L217E ++A098F-S099T-G102A-S103A ++T022A-S078N-G097S-S101Q-S103A-V104I-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271FT022A-S024G-S101G-S103A-V104I-P129E-G159D-S188D-A232V- ++Q245R-N248D-E271F G020R-S078D-G118S-Q245R ++A098Q-S099T-S101A-S103A-V104L ++T022A-G097A-S101N-S103A-V104I-G159D-S188D-L217Q-A232V- ++Q245R-N248D-E271F G020K-S024F-S078N-S128D-T213A-L217E ++T022A-G097A-S101N-S103A-V104I-G159D-S188D-A232V-Q245R- ++ N248D-E271FG020R-S078D-G118S-S188D-Q245R-H249R ++T022A-G097A-S101N-S103A-V104I-P129E-G159D-S188D-A232V- ++Q245R-N248D-E271F T022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245V-N248E++ T022A-S024G-S078N-G097S-S101N-S103A-V104I-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271FT022A-S101G-S103A-V104I-P129E-A158E-S188D-A232V-Q245R- ++ N248D-E271FT022A-S078N-S101N-S103A-V104I-G159D-S188D-A232V-Q245R- ++ N248D-E271FT022A-S024G-N076D-G097S-A098E-S101G-S103A-V104I-G159D- ++S188D-A232V-Q245R-N248D-E271FT022A-N076D-S101G-S103A-V104I-A158E-S188D-A232V-Q245R- ++ N248D-E271FG020K-T022L-S078N-S166D-T213A ++ S101G-S103A-V104L-S128N ++T022A-S101G-S103A-V104I-N116L-S128L-G159D-S188D-T213A- ++A232V-Q245R-N248D-E271FT022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245T-N248D ++T022A-G097A-S101Q-S103A-V104I-S128A-G159D-S188D-A232V- ++Q245R-N248D-E271F ++ S099G-G100S ++T022A-N076D-G097A-S101Q-S103A-V104I-S128A-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271FG020R-N062E-S078D-G118D-P129E-Q245R-N248D-H249R ++T022A-S078N-G097S-S101Q-S103A-V104I-Q109G-S128A-G159D- ++S188D-A232V-Q245R-N248D-E271FT022A-S101N-S103A-V104I-S128A-G159D-S188D-L217Q-A232V- ++Q245R-N248D-E271F T022A-S078N-G097A-S101G-S103A-V104I-S128A-G159D-S188D-++ A232V-Q245R-N248D-E271F T022A-N116L-A158E-S188D ++ S099A-G102A-V104L++ T033S-N116L-A158E-A272D ++T022Q-S101G-S103A-V104I-G159D-S188E-A232T-Q245T-N248E ++S101G-S103A-V104I-N116A-S128N-Y209A-G211Q-A232V ++G020K-T022L-S078N-G118R-S128D-T213A-L217E ++T022A-N076D-G097A-S101Q-S103A-V104I-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271F T022A-S024R-T033S-N116L-G118R-S166D-S188D ++T022L-S024F-G118R-S166D-T213A ++ T022A-S103A-V104I-L111V-S128N ++T022A-N076D-S078N-G097S-S101N-S103A-V104I-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271FT022A-G097A-S101N-S103A-V104I-P129S-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271FT022A-S024G-S078N-S101N-S103A-V104I-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271FT022A-S101G-S103A-V104I-G159D-S188D-A232V-V244A-Q245R- ++N248D-A270V-E271F-A272V T022A-T033S-N116L-G118R-S128I ++T022A-S024G-N076D-S078N-G097A-S101G-S103A-V104I-S128A- ++G159D-S188D-A232V-Q245R-N248D-E271F S024R-S078G-G118S-T180N-Q245R-N248D++ S099G-S103G-V104I ++T022A-S101N-S103A-V104I-S128A-G159D-S188D-A232V-Q245R- ++ N248D-E271FT022A-G097A-S101Q-S103A-V104I-S128A-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271FT022A-S024G-G097S-S101Q-S103A-V104I-S128A-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271F N062E-N116L-A158E ++T022A-S024G-N076D-S101N-S103A-V104I-S128A-G159D-S188D- ++A232V-Q245R-N248D-E271F T022L-S024F-S078N-S166D-T213A-L217E ++T022A-S101G-S103A-V104I-G159E-S188D-A232V-Q245R-N248D- ++ E271FT022A-S101N-S103A-V104I-P129S-G159D-S188D-A232V-Q245R- ++ N248D-E271FT022A-S024G-G097S-S101Q-S103A-V104I-S128A-G159D-S188D- ++A232V-Q245R-N248D-E271FT022A-S024R-N076D-S101G-S103A-V104I-I107V-P129E-G159D- ++S188D-A232V-Q245R-N248D-A270V-E271FS024R-N062E-N116L-G118R-S128I-A158E-S188D ++A016S-S024F-S101G-S103A-V104I-N116L-Y209V-G211Q-A232V ++T022A-S024R-S101G-S103A-V104I-P129E-G159D-S188D-A232V- ++N238Y-Q245R-N248D-E271F-A272VT022A-S024G-N076D-G097A-S101G-S103A-V104I-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271FT022A-N062Q-S101N-S103A-V104I-G159D-S188D-L217Q-A232V- ++Q245R-N248D-E271F S024R-N062E-A158E-S166D-S188D ++T022A-S101G-S103A-V104I-G159D-S188D-A232S-Q245V-N248E ++N062E-N116L-G118R-S128I ++ T033S-N062E-N116L-G118R-A158E-S166D ++G020R-N062E-S078G-G118D-P129E-S188D-Q245R-H249R ++T022A-N076D-S078N-S101N-S103A-V104I-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271FT022A-S078N-S101N-S103A-V104I-S128A-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271F G020K-S166D-T213A ++T022A-S024G-N076D-G097S-S101Q-S103A-V104I-S128A-G159D- ++S188D-L217Q-A232V-Q245R-N248D-E271F T033S-N116L-G118R-A158E-S166D-S188D++ S024R-S078D-G118S-P129E-S188D-N248D ++S024R-N062E-S078G-G118D-G159D-N248D ++T022A-G097A-S101N-S103A-V104I-L124V-P129E-G159D-S188D- ++A232V-Q245R-N248D-E271F T022L-S024F-S078N-S166D-T213A ++G020R-S024R-S078G-G118D-S188D-H249R ++T022A-G097A-S101Q-S103A-V104I-P129E-G159D-S188D-A232V- ++Q245R-N248D-E271F A016S-T022A-V104I-L111V ++T022A-S078N-G097A-S101Q-S103A-V104I-S128A-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271FS024F-T033S-S101G-S103A-V104I-N116L-Y209V-A232V ++T022A-S101G-S103A-V104I-P129E-A158E-G159E-S188D-A232V- ++Q245R-N248D-E271F T022A-N062E-N116L-G118R ++T022A-N076D-G097S-S101G-S103A-V104I-S128A-G159D-S188D- ++A232V-Q245R-N248D-E271F A016S-T022A-S101G-V104I-L111V-L148I ++S024R-N116L-S128I-A158E ++T022A-S024F-S101G-S103A-V104I-N116A-Y209A-A232V ++T022A-S024R-N076D-S101G-S103A-V104I-G159D-S188D-A232V- ++Q245R-N248D-A270V-E271F-A272V N062E-S078D-G118S-G159D-S188D-K235R-Q245R++ T022A-S101Q-S103A-V104I-S128A-P129S-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271FT022A-G097S-S101G-S103A-V104I-S128A-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271F T022A-S103G-V104I-S128N-L148I ++T022A-N076D-S078N-S101G-S103A-V104I-S128A-G159D-S188D- ++A232V-Q245R-N248D-E271FT022A-N076D-S078N-G097S-S101G-S103A-V104I-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271FT022A-S024G-S078N-G097S-S101N-S103A-V104I-S128A-G159D- ++S188D-A232V-Q245R-N248D-E271FT022A-S101N-S103A-V104I-L124V-S128A-P129S-G159D-S188D- ++A232V-Q245R-N248D-E271FT022A-S024F-S101G-S103A-V104I-N116L-Y209V-G211Q-T213A- ++ A232VS024F-G118R-S128D ++ N076D-S101A-S103A-V104I-A232V-Q245R ++T022A-S101Q-S103A-V104I-L124V-S128A-G159D-S188D-A232V- ++Q245R-N248D-E271F T022A-N076D-S101Q-S103A-V104I-S128A-G159D-S188D-L217Q-++ A232V-Q245R-N248D-E271F T033S-S166D ++T022A-S024G-S078N-S101G-S103A-V104I-G159D-S188D-A232V- ++Q245R-N248D-E271F T022A-S078N-G097S-S101N-S103A-V104I-G159D-S188D-A232V-++ Q245R-N248D-E271F A114L ++G020R-S024R-S078D-G118S-P129E-G159D-K237E-N248D-H249R ++G020K-T022L-S024F-G118R-S128D-T213A-L217E ++A016S-S101A-S103A-V104L-S128N-L148I ++T022A-N076D-S078N-S101Q-S103A-V104I-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271FT022A-N076D-S078N-G097S-S101N-S103A-V104I-S128A-G159D- ++S188D-A232V-Q245R-N248D-E271FT022A-G097A-S101N-S103A-V104I-L124V-P129S-G159D-S188D- ++A232V-Q245R-N248D-E271FT022A-S024G-S078N-S101Q-S103A-V104I-P129E-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271FA016S-T022A-S101G-S103A-V104I-N116L-Y209V-A232V ++T022A-G097A-S101N-S103A-V104I-L124V-S128A-P129S-G159D- ++S188D-A232V-Q245R-N248D-E271F T022A-S024R-T033S-G118R-S188D ++T022A-T033S-N116L-G118R-S128I-S188D ++T022A-S024R-T033S-S128I-A158E-S188D ++T022A-S101Q-S103A-V104I-S128A-G159D-S188D-L217Q-A232V- ++Q245R-N248D-E271F A016S-T022A-S103A-L148I ++T022A-N076D-S078N-G097A-S101N-S103A-V104I-S128A-G159D- ++S188D-A232V-Q245R-N248D-E271FN018S-T022A-S024G-S101N-S103A-V104I-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271FT022A-N076D-S078N-G097A-S101Q-S103A-V104I-G159D-S188D- ++A232V-Q245R-N248D-E271FT022A-G097S-S101N-S103A-V104I-S128A-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271FT022A-N076D-S078N-S101N-S103A-V104I-S128A-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271FT022A-S024R-S101G-S103A-V104I-P129E-G159D-S188D-A232V- ++Q245R-N248D-E271F V104L-L111V ++ T066V ++T022A-G097A-S101N-S103A-V104I-S128A-P129S-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271FT022A-S024G-S078N-G097S-S101Q-S103A-V104I-Q109G-G159D- ++S188D-L217Q-A232V-Q245R-N248D-E271F S099T-S101A-S103G ++ T253L ++N076D-S101A-S103A-V104I-P210I-M222S-A232V-Q245R ++ S099G-S101A-G102A ++T022A-G097A-S101N-S103A-V104I-L124V-S128A-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271FT022A-S101G-S103A-V104I-P129E-G159D-S188D-A232V-Q245R- ++N248D-A270V-E271F-A272VT022A-S024G-G097A-S101N-S103A-V104I-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271F S024R-S078D-G118D-S188D-Q245R ++T022A-S103G-V104L-S128N ++ S024R-N116L-G118R ++T022A-N076D-S101G-S103A-V104I-I107V-N123S-P129E-G159D- ++S188D-A232V-N238Y-Q245R-N248D-A270V-E271FT022A-N076D-S078N-S101Q-S103A-V104I-S128A-G159D-S188D- ++A232V-Q245R-N248D-E271FT022A-N076D-S078N-G097S-S101N-S103A-V104I-S128A-G159D- ++S188D-L217Q-A232V-Q245R-N248D-E271FT022A-N043R-N076D-S101G-S103A-V104I-P129E-A158E-G159E- ++S188D-A232V-Q245R-N248D-E271F-A272VT022A-S024G-N076D-S078N-S101N-S103A-V104I-S128A-G159D- ++S188D-A232V-Q245R-N248D-E271F T033S-N062E ++T022A-N076D-S101G-S103A-V104I-A158E-S188D-A232V-Q245R- ++N248D-E271F-A272V A098Q-S099A-G102A ++ G118R-S128I-A158E-S188D ++T022A-S024G-N076D-G097A-S101G-S103A-V104I-S128A-G159D- ++S188D-A232V-Q245R-N248D-E271FT022A-G097S-S101N-S103A-V104I-Q109G-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271F T022A-S103A-V104L-S128N ++T022A-S101N-S103A-V104I-S128A-P129S-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271FT022A-S024G-G097S-S101Q-S103A-V104I-G159D-S188D-A232V- ++Q245R-N248D-E271F T022A-T033S-A158E ++ S024R-S188D ++T022A-S101G-S103A-V104I-G159D-S188D-A232L-Q245V-N248H- ++ E271TS024R-T033S-N062E ++ S024R-T033S-S166D ++T022A-T033S-S101G-S103A-V104I-G118R-G159D-S188D-A232V- ++Q245R-N248D-E271F T022A-S024R-N062E-A158E-S166D-S188D ++T022A-N076D-G097A-S101N-S103A-V104I-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271FT022A-S024G-N076D-G097S-S101Q-S103A-V104I-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271FS024R-S101A-S103A-V104I-S188D-P210I-M222S-A232V-Q245R ++T022A-N076D-G097S-S101N-S103A-V104I-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271F T022L-G118R-S128D-T213A ++T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245V-N248E ++T022A-N076D-G097S-S101Q-S103A-V104I-G159D-S188D-A232V- ++Q245R-N248D-E271F T022A-S024G-N076D-G097A-S101G-S103A-V104I-G159D-S188D-++ A232V-Q245R-N248D-E271FT022A-S024R-S101G-S103A-V104I-P129E-A158E-S188D-A232V- ++Q245R-N248D-E271F S056G ++G020R-N062E-S078D-G118S-P129E-S188D-Q245R-N248D-H249R ++T022A-N076D-G097S-S101Q-S103A-V104I-Q109G-S128A-G159D- ++S188D-A232V-Q245R-N248D-E271F S024R-S078D-G118S-P129E-S188D-Q245R-H249R++ T022A-N076D-G097S-S101Q-S103A-V104I-S128A-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271FT022A-S101Q-S103A-V104I-L124V-P129E-G159D-S188D-A232V- ++Q245R-N248D-E271F-R275HT022A-S024G-S078N-G097A-S101G-S103A-V104I-S128A-G159D- ++S188D-A232V-Q245R-N248D-E271FT022A-S024G-R045S-S101G-S103A-V104I-G159D-S188D-A232V- ++Q245R-N248D-E271F T022A-N076D-S101G-S103A-V104I-P129E-G159D-S188D-A232V-++ Q245R-N248D-A270V-E271F-A272VT022A-S101G-S103A-V104I-L126I-P129E-G159D-S188D-A232V- ++Q245R-N248D-E271F T022A-S024G-N076D-S101N-S103A-V104I-G159D-S188D-L217Q-++ A232V-Q245R-N248D-E271FT022A-N062D-S101G-S103A-V104I-N116L-G159D-S188D-T213A- ++A232V-Q245R-N248D-E271FT022A-S024G-N076D-S078N-G097S-S101Q-S103A-V104I-G159D- ++S188D-A232V-Q245R-N248D-E271FT022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D- ++A270V-E271F-A272V S024R-N062E-S078G-G118D-P129E-Q245R-H249R ++T022A-S101G-S103A-S128N ++ G020R-S024R-S078G-G118D-P129E-N248D-H249R ++N116L-G118R-P129E-S188D ++T022A-S101N-S103A-V104I-G159D-S188D-L217Q-A232V-Q245R- ++ N248D-E271FT022A-S101G-S103A-V104I-L126I-S128A-P129S-G159D-S188D- ++A232V-Q245R-N248D-E271FT022A-N076D-S101G-S103A-V104I-Q109G-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271FT022A-S024G-G097A-S101Q-S103A-V104I-S128A-G159D-S188D- ++A232V-Q245R-N248D-E271F G020R-S078D-G118S-P129E-G159D-S188D ++T022A-T033S-G118R-S166D-S188D ++ S078G-G118D-P129E-G159D-SI88D ++T022A-S024G-G097S-S101N-S103A-V104I-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271FT022A-N076D-S101Q-S103A-V104I-G159D-S188D-L217Q-A232V- ++Q245R-N248D-E271F T022A-S024R-S101G-S103A-V104I-P129E-S188D-A232V-Q245R-++ N248D-E271F-A272V T022A-S103A-V104I-L111V ++T022A-S024G-S078N-S101N-S103A-V104I-G159D-S188D-A232V- ++Q245R-N248D-E271F T022A-N076D-S101N-S103A-V104I-S128A-G159D-S188D-L217Q-++ A232V-Q245R-N248D-E271FT022A-N076D-A085T-S101G-S103A-V104I-A158E-S188D-A232V- ++Q245R-N248D-E271F T022A-S024G-S078N-E089G-S101Q-S103A-V104I-G159D-S188D-++ L217Q-A232V-Q245R-N248D-E271FT022A-S101Q-S103A-V104I-Q109N-G159D-S188D-A232V-Q245R- ++ N248D-E271FT022A-N076D-S078N-S101Q-S103A-V104I-G159D-S188D-A232V- ++Q245R-N248D-E271F T022A-S101N-S103A-V104I-L124V-S128A-P129E-G159D-S188D-++ L217Q-A232V-Q245R-N248D-E271FT022A-S024G-G097A-S101Q-S103A-V104I-G159D-S188D-A232V- ++Q245R-N248D-E271F T022A-G097A-S101Q-S103A-V104I-S128A-P129E-G159D-S188D-++ A232V-Q245R-N248D-E271FT022A-S101G-S103A-V104I-P129E-G159D-S188D-A232V-Q245R- ++N248D-E271F-A272V S101G-S103A ++ L196S ++T022A-S024G-S078N-S101G-S103A-V104I-S128A-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271FT022A-G097A-S101G-S103A-V104I-G159D-S188D-L217Q-A232V- ++Q245R-N248D-E271F T022A-S024G-N076D-S078N-S101N-S103A-V104I-Q109N-S128A-++ 0159D-S188D-A232V-Q245R-N248D-E271FT022A-S078N-S101G-S103A-V104I-G159D-S188D-A232V-Q245R- ++ N248D-E271FT022A-G097A-S101Q-S103A-V104I-L124V-S128A-G159D-S188D- ++A232V-Q245R-N248D-E271F E271S ++ A098Q-S099T-G100S-S101A ++T022A-S024G-N076D-G097A-S101N-S103A-V104I-Q109G-G159D- ++S188D-L217Q-A232V-Q245R-N248D-E271FT022A-S101Q-S103A-V104I-Q109N-S128A-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271FT022A-G097A-S101Q-S103A-V104I-Q109N-G159D-S188D-A232V- ++Q245R-N248D-E271F T022A-S101Q-S103A-V104I-S128A-G159D-S188D-A232V-Q245R-++ N248D-E271F T022A-S024G-S078N-G097A-S101Q-S103A-V104I-S128A-G159D- ++S188D-L217Q-A232V-Q245R-N248D-E271FT022A-G097S-S101N-S103A-V104I-G159D-S188D-L217Q-A232V- ++Q245R-N248D-E271F T022A-S024R-N076D-S101G-S103A-V104I-P129E-G159E-S188D-++ A232V-Q245R-N248D-E271FR019H-T022A-N043R-N076D-S101G-S103A-V104I-P129E-S188D- ++A232V-Q245R-N248D-E271F-A272VT022A-S024G-N076D-S078N-G097S-S101Q-S103A-V104I-S128A- ++G159D-S188D-A232V-Q245R-N248D-E271F T022L-S078N-S166D-T213A ++G020R-S024R-S078D-G118D-Q245R-N248D ++ N269K ++T022A-S101N-S103A-V104I-Q109G-G159D-S188D-A232V-Q245R- ++ N248D-E271FG097P-V139A ++ T022A-S101Q-S103A-V104I-S128A-P129S-G159D-S188D-A232V-Q245R-N248D-E271F ++S024F-S101G-S103A-V104I-N116A-S128N-Y209V-G211Q-A232V ++T022A-S101N-S103A-V104I-L124V-S128A-G159D-S188D-L217Q-A232V-Q245R-N248D-E271F ++T022A-S024G-G097A-S101G-S103A-V104I-S128A-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271F N062E-S078G-G118D-G159D-S188D-Q245R-H249R++ T022A-S024G-S101N-S103A-V104I-S128A-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271F N062E-S078D-G118D-P129E-G159D-Q245R-H249R ++T022A-G097A-S101N-S103A-V104I-S128A-G159D-S188D-A232V- ++Q245R-N248D-E271F T022A-S078N-S101Q-S103A-V104I-G159D-S188D-L217Q-A232V-++ Q245R-N248D-E271F S128I-S188D ++T022A-S024G-N076D-G097S-S101Q-S103A-V104I-Q109N-S128A- ++G159D-S188D-L217Q-A232V-Q245R-N248D-E271FT022A-N076D-S078N-S101G-S103A-V104I-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271F S166D-L217E ++T022A-S024G-N076D-G097S-S101G-S103A-V104I-G159D-S188D- ++A232V-Q245R-N248D-E271F A098Q-S099G-S101A ++T022A-S024R-S101G-S103A-V104I-G159D-S188D-A232V-Q245R- ++N248D-A270V-E271F-A272VT022A-V068A-S101G-S103A-V104I-G118R-G159D-S188D-T213A- ++A232V-Q245R-N248D-E271FT022A-S024G-S078N-S101Q-S103A-V104I-Q109N-G159D-S188D- ++A232V-Q245R-N248D-E271FT022A-N076D-S101N-S103A-V104I-S128A-G159D-S188D-A232V- ++Q245R-N248D-E271F T022A-S101G-S103A-V104I-P129S-G159D-S188D-L217Q-A232V-++ Q245R-N248D-E271FT022A-S024G-N076D-G097S-S101Q-S103A-V104I-Q109N-G159D- ++S188D-A232V-Q245R-N248D-E271FT022A-S101Q-S103A-V104I-S128A-P129E-G159D-S188D-A232V- ++Q245R-N248D-E271F S128I-A158E-S166D ++T022A-G097A-S101N-S103A-V104I-L124V-S128A-P129S-G159D- ++S188D-L217Q-A232V-Q245R-N248D-E271FT022A-S024R-N076D-S101G-S103A-V104I-P129E-A158E-S188D- ++A232V-Q245R-N248D-E271F S024R-N062E-S078D-G118D ++T022A-G097A-S101N-S103A-V104I-L126I-G159D-S188D-A232V- ++Q245R-N248D-E271F T022A-S101G-S103A-V104I-S128A-P129S-G159D-S188D-A232V-++ Q245R-N248D-E271F S024F-S078N-G118R-N204K-T213A-L217E ++G020R-N062E-S078D-G118D-G159D-N248D ++T022A-S024G-N076D-G097A-S101Q-S103A-V104I-G159D-S188D- ++A232V-Q245R-N248D-E271FT022A-G097A-S101Q-S103A-V104I-L124V-P129E-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271FT022A-S101G-S103A-V104I-S128A-G159D-S188D-A232V-Q245R- ++ N248D-E271FG097P-S099G-S101G-V104I ++ S024R-N062E-S078D-G118D-N248D ++T022A-S101Q-S103A-V104I-G159D-S188D-L217Q-A232V-Q245R- ++ N248D-E271FT022A-S101G-S103A-V104I-L124V-S128A-P129E-G159D-S188D- ++A232V-Q245R-N248D-E271F S128I ++ G020K-T022L-S128D-T213A-L217E ++G020K-G118R-S166D-T213A ++ A016S-T022A-L111V ++S078D-G118D-P129E-N248D-H249R ++T022A-N076D-S101G-S103A-V104I-G159D-S188D-A232V-Q245R- ++ N248D-E271FS024R-S128I-A158E-S166D-A194T ++T022A-N043R-S101G-S103A-V104I-A158E-G159E-S188D-A232V- ++Q245R-N248D-E271F T022A-N076S-S101G-S103A-V104I-G159D-S188D-A232V-N238Y-++ Q245R-N248D-E271F-A272VT022A-G097A-S101Q-S103A-V104I-P129S-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271FI022A-S101N-S103A-V104I-Q109N-G159D-S188D-A232V-Q245R- ++ N248D-E271FT022A-S024R-S101G-S103A-V104I-P129E-S188D-A232V-Q245R- ++ N248D-E271FG020R-S078D-G118D-P129E-S188D ++ T022A-S101G-S103A-V104I ++T022A-G097A-S101N-S103A-V104I-S128A-G159D-R186H-S188D- ++A232V-Q245R-N248D-E271FT022A-S101G-S103A-V104I-L124V-S128A-P129S-G159D-S188D- ++A232V-Q245R-N248D-E271F G020K-S024F-S078N-G118R-S128D-S166D-T213A ++G020R-N062E-S078D-G118D-G159D-S188D ++T022A-G097S-S101Q-S103A-V104I-G159D-S188D-L217Q-A232V- ++Q245R-N248D-E271F T022A-S024G-S078N-G097A-S101N-S103A-V104I-S128A-G159D-++ S188D-A232V-Q245R-N248D-E271F T033S-N116L-G118R-S128I-S166D ++T022A-S024F-S101G-S103A-V104I-N116L-Y209A-A232V ++T022L-S024F-S078N-G118R-T213A-L217E ++T022A-N076D-G097A-S101Q-S103A-V104I-S128A-G159D-S188D- ++A232V-Q245R-N248D-E271FT022A-N076D-S078N-S101G-S103A-V104I-G159D-S188D-A232V- ++Q245R-N248D-E271F T022A-S024G-N076D-S078N-G097S-S101N-S103A-V104I-G159D-++ S188D-A232V-Q245R-N248D-E271FT022A-N076D-S101N-S103A-V104I-Q109G-S128A-G159D-S188D- ++A232V-Q245R-N248D-E271FT022A-S024G-S101Q-S103A-V104I-Q109N-S128A-G159D-S188D- ++A232V-Q245R-N248D-E271F N062E-S078G-G118S-P129E-S188D ++G020R-N062E-S078D-G118D-G159D-Q245R-H249R ++T022A-G097S-S103A-V104I-Q109N-S128A-G159D-S188D-A232V- ++Q245R-N248D-E271F T022A-S024R-T033S-N062E-N116L-S166D-S188D ++G020R-S078G-G118S-S188D-Q245R-N248D-H249R ++T022A-G097A-S101G-S103A-V104I-S128A-G159D-S188D-A232V- ++Q245R-N248D-E271F G020R-N062E-S078G-G118D-Q245R-N248D-H249R ++T022A-S024G-G097A-S101Q-S103A-V104I-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271FT022A-N076D-S078N-G097S-S101Q-S103A-V104I-G159D-S188D- ++A232V-Q245R-N248D-E271FT022A-S078N-S101G-S103A-S128A-G159D-S188D-L217Q-A232V- ++Q245R-N248D-E271F T022A-S078N-S101Q-S103A-V104I-Q109N-G159D-S188D-A232V-++ Q245R-N248D-E271F G020K-T022L-S024F-S078N-S166D-T213A ++A085T-G097P-A098Q ++ N062E-S078G-G118D-P129E-G159D-Q245R-N248D-H249R ++T022A-S024G-N076S-S078N-G097A-S101N-S103A-V104I-S128A- ++G159D-S188D-A232V-Q245R-N248D-E271FT022A-S078N-S101Q-S103A-V104I-S128A-G159D-S188D-A232V- ++Q245R-N248D-E271F Q012R-T022A-S101G-S103A-V104I-N116L-Y209A-T213A-A232V++ T022A-N076D-G097S-S101N-S103A-V104I-S128A-G159D-S188D- ++A232V-Q245R-N248D-E271FT022A-S024G-S101G-S103A-V104I-G159D-S188D-A232V-Q245R- ++ N248D-E271FN116L-A158E-S166D-S188D ++T022A-S024R-N076D-S101G-S103A-V104I-A158E-G159E-S188D- ++A232V-Q245R-N248D-E271FT022A-S024G-N076D-S078N-G097A-S101Q-S103A-V104I-Q109N- ++G159D-S188D-A232V-Q245R-N248D-E271F S024R-T033S-S128I-A158E ++S024R-N116L-G118R-S128I-S188D ++T022A-N043R-S101G-S103A-V104I-P129E-S188D-A232V-Q245R- ++ N248D-E271FN062E-S078D-G118D-S188D-H249R ++G020R-N062E-S078D-G118S-P129E-G159D-S188D ++G020K-T022L-S078D-G118D-P129E-N248D-H249R ++T022A-S024R-N043R-N076D-S101G-S103A-V104I-A158E-S188D- ++A232V-Q245R-N248D-E271FT022A-N076D-G097A-S101G-S103A-V104I-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271F ++T022A-G097A-S101N-S103A-V104I-L124V-P129S-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271FT022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245V-N248D ++N062E-S078D-G118S-P129E-S188D-Q245R ++S024R-N062E-G118R-S128I-A158E-S188D ++T022A-S101N-S103A-V104I-P129E-G159D-S188D-A232V-Q245R- ++ N248D-E271FG020K-T022L-S078N-S128D-L217E ++ T022A-N062E-N116L-A158E-S188D ++T022A-S101A-V104I-L111V ++ S166D-T213A ++T022A-G097A-S101N-S103A-V104I-L124V-S128A-P129E-G159D- ++S188D-A232V-Q245R-N248D-E271F T022L-S024F-S166D-T213A ++T022A-N076D-S101Q-S103A-V104I-G159D-S188D-A232V-Q245R- ++ N248D-E271FT022A-S024R-S101G-S103A-V104I-P129E-G159D-S188D-A232V- ++N238Y-Q245R-N248D-A270V-E271FT022A-G097S-S101Q-S103A-V104I-Q109G-S128A-G159D-S188D- ++A232V-Q245R-N248D-E271F S024R-S128I-A158E-S166D ++N062E-S078D-G118S-N248D-H249R ++T022A-S024G-S101Q-S103A-V104I-Q109G-G159D-S188D-A232V- ++Q245R-N248D-E271F T022A-S024G-N076D-S078N-G097S-S101Q-S103A-V104I-G159D-++ S188D-L217Q-A232V-Q245R-N248D-E271F T022L-S024F-S078N-S166D ++T022A-S101G-S103A-V104I-P129E-A158E-S188D-A232V-Q245R- ++N248D-E271F-A272V S078D-G118S-P129E-N243D-H249R ++T022A-S024G-N076D-S078N-S101N-S103A-V104I-S128A-G159D- ++S188D-L217Q-A232V-Q245R-N248D-E271FT022A-S078N-S101G-S103A-V104I-S128A-G159D-S188D-A232V- ++Q245R-N248D-E271F T022A-S024R-N062E-G118R ++T022A-N076D-S101G-S103A-V104I-P129E-S188D-A232V-Q245R- ++N248D-E271F-A272V T022A-S078N-S101Q-S103A-V104I-S128A-G159D-S188D-L217Q-++ A232V-Q245R-N248D-E271F S024R-S078G-G118S-Q245R ++T022A-S024G-N076D-S078N-S101G-S103A-V104I-G159D-S188D- ++A232V-Q245R-N248D-E271F-A272TT022A-S024G-N076D-S078N-G097A-S101Q-S103A-V104I-S128A- ++G159D-S188D-L217Q-A232V-Q245R-N248D-E271FT022A-G097A-S101G-S103A-V104I-P129S-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271FT022A-S024G-S078N-G097S-S101G-S103A-V104I-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271FT022A-G097A-S101G-S103A-V104I-L124V-S128A-P129E-G159D- ++S188D-A232V-Q245R-N248D-E271F S078N-S166D-L217E ++T022A-N076D-S101G-S103A-V104I-I107V-P129E-G159D-S188D- ++A232V-N238Y-Q245R-N248D-A270V-E271F-A272V Q236L ++S078D-G118D-P129E-H249R ++T022A-S101Q-S103A-V104I-G159D-S188D-A232V-Q245R-N248D- ++ E271FT022A-S101A-S103A-V104I-L111V ++ T022L-S024F-S078N-S128D-T213A ++T022A-S024G-N076D-G097A-S101N-S103A-V104I-S128A-G159D- ++S188D-A232V-Q245R-N248D-E271F G020K-S024F-S078N-G118R-S128D-L217E ++T022A-S101G-S103A-V104I-G159E-S188D-A232V-Q245R-N248D- ++ E271F-A272VT022A-N076D-S101G-S103A-V104I-P129E-G159D-S188D-A232V- ++Q245R-N248D-E271F T022A-S024G-S078N-S101G-S103A-V104I-G159D-S188D-L217Q-++ A232V-Q245R-N248D-E271FT022A-G097A-S101N-S103A-V104I-P129E-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271FT022A-N076D-S101N-S103A-V104I-Q109N-S128A-G159D-S188D- ++A232V-Q245R-N248D-E271FT022A-N043R-N076D-S101G-S103A-V104I-A158E-G159E-S188D- ++A232V-Q245R-N248D-E271F-A272V G020K-S128D-T213A-L217E ++S103A-V104I-L111V-S128N ++T022A-G097S-S101N-S103A-V104I-Q109N-S128A-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271FT022A-S024G-S101Q-S103A-V104I-G159D-S188D-L217Q-A232V- ++Q245R-N248D-E271F T022A-N076D-S101G-S103A-V104I-I107V-G159D-S188D-A232V-++ Q245R-N248D-A270V-E271F-A272V V104I-L111V ++T022A-N062Q-S101Q-S103A-V104I-P129E-G159D-S188D-A232V- ++Q245R-N248D-E271F T022A-G097A-S101N-S103A-V104I-L124V-G159D-S188D-A232V-++ Q245R-N248D-E271F A016S-S101A-L111V-S128N ++T022A-S024G-N076D-S078N-G097A-S101G-S103A-V104I-G159D- ++S188D-L217Q-A232V-Q245R-N248D-E271FT022A-S101G-S103A-V104I-G159D-S188E-A232L-Q245V-N248E ++T022A-N076D-G097A-S101N-S103A-V104I-S128A-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271FT022A-S024G-N076D-G097S-S101N-S103A-V104I-Q109G-G159D- ++S188D-A232V-Q245R-N248D-E271FT022A-S101G-S103A-V104I-I107V-G159D-S188D-A232V-Q245R- ++ N248D-E271FT022A-N076D-G097S-S101G-S103A-V104I-G159D-S188D-A232V- ++Q245R-N248D-E271F T022A-S101G-S103A-V104I-S128L-G159D-S188D-A232V-Q245R-++ N248D-E271F-A272DT022A-S101Q-S103A-V104I-L126I-P129S-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271FT022A-S024G-N076D-S078N-S101Q-S103A-V104I-S128A-G159D- ++S188D-L217Q-A232V-Q245R-N248D-E271FA016S-T022A-S101G-S103A-V104I-N116L-Y209V-G211Q-T213A- ++ A232VT022A-S101Q-S103A-V104I-L124V-S128A-P129E-G159D-S188D- ++A232V-Q245R-N248D-E271FT022A-S024G-N076D-G097A-S101N-S103A-V104I-S128A-G159D- ++S188D-L217Q-A232V-Q245R-N248D-E271FT022A-S101N-S103A-V104I-Q109N-S128A-G159D-S188D-A232V- ++Q245R-N248D-E271F S101G-S103A-V104I-N116A-S128N-Y209V-G211Q-A232V ++S024R-S078G-G118S-G159D-S188D-Q245R-H249R ++ T022A-S103G-S128N-L148I ++T022A-S101Q-S103A-V104I-S128A-P129E-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271FT022A-N076D-G097S-S101Q-S103A-V104I-Q109N-G159D-S188D- ++A232V-Q245R-N248D-E271F G020R-S024R-S078D-G118S-N248D ++T022Q-S101G-S103A-V104I-G159D-S188D-A232S-Q245V-N248D- ++ E271TS078G-G118D-S188D-Q245R-N248D ++T022A-N076D-S078N-G097S-S101N-S103A-V104I-Q109G-G159D- ++S188D-A232V-Q245R-N248D-E271F G118R-S128D-S166D-L217E ++G020R-S078G-G118S-P129E-Q245R-H249R ++A016S-S024F-S101G-S103A-V104I-N116A-S128N-Y209V-T213A- ++ A232VT022A-S078N-S101Q-S103A-V104I-R145K-G159D-S188D-L217Q- ++A232V-Q245R-N248D-E271FT022A-N043R-N076D-S101G-S103A-V104I-P129E-A158E-S188D- ++A232V-Q245R-N248D-E271FT022A-G097A-S101Q-S103A-V104I-L126I-P129S-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271FT022A-G097A-S101N-S103A-V104I-Q109N-G159D-S188D-A232V- ++Q245R-N248D-E271F T022A-S024G-S078N-S101N-S103A-V104I-S128A-G159D-S188D-++ A232V-Q245R-N248D-E271FT022A-S024R-N076D-S101G-S103A-V104I-P129E-S188D-A232V- ++Q245R-N248D-E271F-A272V T022L-S078N-T213A ++T022A-N076D-S078N-G097A-S101G-S103A-V104I-G159D-S188D- ++A232V-Q245R-N248D-E271FT022A-S078N-S101G-S103A-V104I-Q109G-S128A-G159D-S188D- ++A232V-Q245R-N248D-E271F N062E-S078G-G118S-P129E-G159D-Q245R-H249R ++A016S-S101G-V104I-S128N-L148I ++ T022A-G118R-S188D ++T022A-G097A-S101Q-S103A-V104I-G159D-S188D-A232V-Q245R- ++ N248D-E271FG020K-S024F-G118R-S128D-T213A ++T022A-S101Q-S103A-V104I-P129E-G159D-S188D-A232V-Q245R- ++ N248D-E271FG020K-T022L-S024F-S078N-S128D-T213A-L217E ++T022A-S024G-S078N-G097A-S101Q-S103A-V104I-Q109G-S128A- ++G159D-S188D-A232V-Q245R-N248D-E271FA016S-S101G-S103A-V104I-N116L-Y209V-G211Q-A232V ++T022A-S024G-G097A-S101Q-S103A-V104I-S128A-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271FT022A-S101N-S103A-V104I-L124V-S128A-P129S-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271F S099G-G100S-S103A ++T022Q-S101G-S103A-V104I-S166D-S188E-A232T-Q245T-N248E ++T022A-S024G-N076D-S078N-G097S-S101N-S103A-V104I-Q109N- ++G159D-S188D-A232V-Q245R-N248D-E271F A098F-S099T-S103A-V104I ++G020K-T022L-N062E-S078D-G118D-P129E-Q245R-N248D ++T022L-S078N-G118R-S128D ++T022A-S101G-S103A-V104I-N116L-Y209A-G211Q-T213A-A232V ++T022A-S101Q-S103A-V104I-L126I-S128A-P129S-G159D-S188D- ++L217Q-A232V-Q245R-N248D-E271FG023A-S024F-S101G-S103A-V104I-N116A-Y209V-A232V ++T022A-S024R-N116L-G118R-S166D ++S024R-N062E-S078D-G118D-P129E-G159D-Q245R-N248D-H249R ++G020K-T022L-S024F-S078N-G118R-S128D-T213A ++T022A-S024G-N076D-S078N-G097A-S101Q-S103A-V104I-S128A- ++G159D-S188D-A232V-Q245R-N248D-E271FT022A-S024G-S101N-S103A-V104I-S128A-G159D-S188D-A232V- ++Q245R-N248D-E271F-T274I G097P-S099A-V104L ++ G097P-S099A-S103A ++T022A-S024G-S078N-G097S-S101Q-S103A-V104I-G159D-S188D- ++A232V-Q245R-N248D-E271F T022A-V104L-L148I ++T022A-S024G-G097A-S101N-S103A-V104I-S128A-G159D-S188D- ++A232V-Q245R-N248D-E271FT022A-S024R-S101G-S103A-V104I-P129E-G159S-S188D-A232V- ++Q245R-N248D-E271F S103G ++T022A-S024G-N076D-S078N-S101G-S103A-V104I-Q109G-G159D- ++S188D-A232V-Q245R-N248D-E271FT022A-S024G-N076D-G097S-S101G-S103A-V104I-G159D-S188D- +L217Q-A232V-Q245R-N248D-E271F G020K-T022L-S078N-G118R-S128D-T213A +S024R-S078G-G118S-P129E-S188D-Q245R-H249R +T022A-N076D-S101G-S103A-V104I-G159D-S188D-L217Q-A232V- +Q245R-N248D-E271FT022A-S101G-S103A-V104I-S128A-G159D-S188D-L217Q-A232V- +Q245R-N248D-E271FT022A-S024G-G097S-S101Q-S103A-V104I-Q109G-G159D-S188D- +A232V-Q245R-N248D-E271FT022A-N076D-S101N-S103A-V104I-G159D-S188D-A232V-Q245R- + N248D-E271FT033S-G118R-S128I-A158E + 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V104L +S024R-T033S-G118R-S128I-A158E-S166D +N062E-S078D-G118S-P129E-G159D-Q245V-N248E-E271T +S024R-T033S-G118R-S128I-S166D + A016S-S101G-S103G-V104L +S078N-S128D-L217E + A016S-T022A-S101A-V104I-L111V + A016M +S099T-G102A-S103A-V104I + T022A-S101A-V104I-L111V-S128N +T022A-S024R-N043R-N076D-S078P-S101G-S103A-V104I-P129E- +G159E-S188D-A232V-Q245R-N248D-E271F T022L-S078N-S128D + V051E +N062E-S078D-G118S-P129E-G159D +T022A-S101N-S103A-V104I-L124T-P129S-G159D-S188D-L217Q- +A232V-Q245R-N248D-E271F A015H +G020R-S024R-N062E-S078D-G118S-G159D-S188D-Q245R-H249R +G020K-S024F-G118R-S128D-S166D-T213A-L217E +A016S-T022A-S101G-S103G-V104I + P014N +T022A-S101Q-S103A-V104I-L124T-S128A-P129E-G159D-S188D- +A232V-Q245R-N248D-E271FT022A-G097A-S101G-S103A-V104I-Q109N-G159D-S188D-A232V- +Q245R-N248D-E271FT022A-S024R-S101G-S103A-V104I-G159D-S188D-A232V-N238Y- +Q245R-N248D-E271F G097P-A098Q-S101A-G102A + T033S-A158E-S166D-S188D +I035C + T033S-S128I-A158E-S188D + I008S + S078N-T213A + V028T-A215V +G020K-T022L-S078N-S128D-S166D-T213A-L217E + G146C-Q236L + G020K +S101G-S103A-V104I-N116A-S128N-Y209V-T213A-A232V +S024R-S078D-G118D-G159D-K237E + S024R-T033S-N116L-S128I +T022A-S024R-S101G-S103A-V104I-I107V-G159D-S188D-A232V- +N238Y-Q245R-N248D-E271F A048G + S212D + N062E-N116L-A158E-S188D +T022A-S024F-S101G-S103A-V104I-N116L-G118V-Y209A-T213A- + A232VS024F-S078N-G118R-S128D-S166D-T213A-L217E + A098F-S101G + S132K +T022A-S101G-S103A-V104I-N116A-G118V-Y209V-T213A-A232V +T022A-T033S-S101G-S103A-V104I-N116L-Y209A-G211Q-A232V +G097P-A098Q-S099T-S101A-G102A-V104I + G097P-S099T-S101A-S103A +A016S-T022A-S024F-T033S-S101G-S103A-V104I-N116L-S128N- + Y209V-A232VA098Q-S099T-S101G-V104I +G020K-T022L-S024F-G118R-S128D-S166D-T213A-L217E +G097P-A098Q-S099G-S101G + G097P-A098Q-S099A-G100S +T033S-N062E-G118R-S166D-S188D +A016S-T022A-S024F-S101G-S103A-V104I-N116L-G118V-Y209V- + T213A-A232VN062E-S078G-G118S-P129E-S188D-Q245R-H249R +S024F-T033S-S101G-S103A-V104I-N116L-S128N-Y209V-T213A- + A232VG097P-S099G-S101G-S103A + A098F-G102A-V104I + S078D-G118S-Q245R-N248D +G097P-A098Q-S099A-G102A + G097P-A098Q-G100S-S103A +T022A-S024R-N062E-S101G-S103A-V104I-P129E-S188D-A232V- +Q245R-N248D-E271F S078G-G118D-Q245R-H249R + G097P-S099A-G100S-S101A +G097P-S099G-S103G + G020K-S024F-S128D-S166D-T213A-L217E + I008N +N269A + A088T + T038S +A016S-T022A-S101G-S103A-V104I-N116A-G118V-Y209A-T213A- + A232VG097P-A098F-S103A-V104I + A098F-S099A +T022A-N076D-S101G-S103A-V104I-I107V-G159D-S188D-A232V- +N238Y-Q245R-N248D-E271F-A272V A098Q-S099T-G100S-V104L +G097P-S099T-V104L + A098Q-S099T-S103A-V104I + A016S-T022A-S103G-S128N +T022A-T033S-S166D-S188D + G097P-G100S-S103A +T022L-S024F-S078N-T213A-A232T + A013S + G097P-S101A-G102A-S103A +A016S-T022A-S101A-V104L-L111V-S128N + G097P-A098Q-S099T-G102A +S024R-S078G-G118D-N248D + G020K-S078N-S128D-S166D-T213A-L217E +G020R-S078G-G118D-N248D + A085S + A016S-T022A-S128N +S024F-S078N-G118R-S128D-S166D-L217E + A098Q-S099A-G100S-V104L +S024R-S078D-G118D-G159D-N248D + A098Q-S103A + N062E-A158E-S166D +G020K-S024F-G118R-S128D-S166D-L217E + G097P-A098F + G080F +G097P-S099A-S103A-V104I + T022A-S101A-L111V-S128N-L148I +A016S-S101G-S103A-V104I +T022A-N062Q-S101Q-S103A-V104I-L124V-P129S-G159D-S188D- +A232V-Q245R-N248D-E271F G097P-A098F-S099T-S101G-V104I + A073Q + K251C +A016S-T022A-S101A-S103A-V104L-L148I + G097P-A098Q-G102A-S103G +G020R-S078G-G118D-Q245R-N248D + S099A-G100S-S103A + T038A +T022A-S024R-S101G-S103A-V104I-G159D-S188D-A232V-N238Y- +Q245R-N248D-A270V-E271F-A272V T022L-S078N-S128D-T213A-L217E +T022A-S024R-N043R-N076D-S101G-S103A-V104I-P129E-G159E- +S188D-A232V-Q245R-N248D-E271F N116L-S128I-A158E-S166D +A016S-T022A-T033S-S101G-S103A-V104I-N116L-Y209A-P210Q- + T213A-A232VS049V-V199I + T022A-S103G-V104I-L148I + A016S-S101G-V104L +A098F-S099T-S101G-G102A-V104I + T022A-A158E-S166D-S188D +A016S-T022A-T033S-S101G-S103A-V104I-N116L-S128N-Y209V- + G211Q-A232VT022A-S024G-N076D-G097A-S101G-S103A-V104I-Q109G-G159D- +S188D-L217Q-A232V-Q245R-N248D-E271FA016S-S024F-T033S-S101G-S103A-V104I-N116A-Y209V-T213A- + A232VN062E-N116L-G118R-S128I-A158E-S188D + N062E-S128I-S188D + G097P +T022A-S024F-T033S-S101G-S103A-V104I-N116A-S128N-Y209A- +G211Q-T213A-A232V S024R-S078G-G118S-P129E-Q245R-H249R + S049D +G097P-A098Q-S099T-G100S + T022A-S024R-T033S-N116L-G118R-S128I-S188D +T022A-T033S-S101G-S103A-V104I-N116A-Y209A-T213A-A232V +T022L-S078N-G118R-S128D-S166D-L217E + S078G-G118D-P129E-N248D +S099A-V104L + T022A-N043R-S101G-S103A-V104I-A158E-S188D-A232V-Q245R- +N248D-E271F A098Q-V104L + T022A-T033S-G118R-S128I-S166D-S188D +G097P-S099T-S101A + W006Y +S024R-N062E-S078G-G118D-P129E-S188D-Q245R-H249R +S024R-S078D-G118D-Q245R-N248D +T022A-N076D-S101G-S103A-V104I-I107V-P129E-G159D-S188D- +A232V-N238Y-Q245R-N248D-E271FT022A-S101N-S103A-V104I-L124V-L126I-S128A-P129E-G159D- +S188D-A232V-Q245R-N248D-E271F B015T +G097P-S099T-S101G-G102A-S103A-V104I + A016S-V104L-L111V-S128N +G097P-A098F-S099T + S099G + S049I + A016S-S103G-V104L-L148I +N062E-S078G-G118D-S188D-N248D +G020R-N062E-S078D-G118S-G159D-S188D-K235R-N248D + T022A-S024R-N116L +S024R-T033S-S128I + N062E-S078D-G118D-Q245R-N248D-H249R +A098F-S099A-S103A-V104I + V004Y + T022L-S024F-S078N-T213A +A016S-S101G-S103A-V104I-N116L-G118V-Y209A-G211Q-A232V +G097P-A098Q-G102A-S103G-V104I + S078G-G118S-H249R +S024F-S101G-S103A-V104I-N116A-S128N-Y209V-A232V + G097P-V104I +S024F-T033S-S101G-S103A-V104I-N116A-S128N-Y209A-T213A- + A232VA098Q-S099G-V104L + N062E-S078D-G118D-P129E + L196K +S078G-G118D-N248D-H249R + S087M +T022A-S101G-S103A-V104I-Q109G-S128A-G159D-S188D-L217Q- +A232V-Q245R-N248D-E271F S216N-T253C + G097P-A098Q-S101G-G102A-V104I +G020K-T022L-G118R + A016S-T022A-S101A-S103G-V104I-L111V +A098F-S099A-G102A + T033S-N062E-G118R-S128I-A158E + A098Q-S099T-S101A +G020R-N062E-S078D-G118S-P129E-G159D-S188D-Q245R-N248D + V011L +S101A-L148I + A098F-S099A-S101A + F050K +N062E-S078G-G118S-G159D-S188D-N248D + V051A +T022A-T033S-G118R-S128I-A158E-S166D + V051A-S212D + P014W + Q012E +T022A-T033S-S128I-A158E-S188D +T022A-S024R-N062E-N076D-S101G-S103A-V104I-P129E-S188D- +A232V-Q245R-N248D-E271F G097P-S103A + S078G-G118D +G020R-S024R-N062E-S078G-G118D-S188D-Q245R-H249R +G097P-A098Q-G102A-V104L +T022A-S101N-S103A-V104I-L124V-S128A-G159D-S188D-A232V- +Q245R-N248D-E271F A098Q-S099T-S101A-G102A-S103A-V104I +A016S-L111V-S128N + T022A-N062E-N116L-S128I-A158E + G080C +S024R-S078D-G118S + G097P-A098F-S099G +A016S-S024F-T033S-S101G-S103A-V104I-N116L-S128N-Y209V- + T213A-A232VG097P-S099G-G100S-S103A + S212A +S024R-N062E-S078D-G118S-P129E-G159D-S188D-N248D-H249R +T022A-S103G-V104L-S128N-L148I +T022A-S101N-S103A-V104I-L124T-S128A-P129S-G159D-S188D- +A232V-Q245R-N248D-E271F A016S-T022A-S103G-V104L-S128N-L148I +G097P-A098Q-S101A-V104I + A016S-S101A-V104I + V026D +G097P-S101A-G102A-S103A-V104I +A016S-T022A-S024F-T033S-S101G-S103A-V104I-N116L-S128N- +Y209A-T213A-A232V T033S-A158E-S188D-A272D + A048V + G020K-T022L-S078N +G020K-G118R-T213A + T022A-S103A-V104L-L148I +T022A-N062E-S128I-A158E-S166D + G097P-A098Q-S099G-G100S-V104I + S049V +G097P-A098Q-S099T-S101A-S103G + G097P-A098F-S099A-S101A-V104I +S128I-A158E-S166D-S188D + S128D-S166D + S099G-G102A-V104I +T022A-S166D + A016S-T022A-S101G-S103A-V104I-N116L-Y209V-T213A-A232V +T022A-T033S-S101G-S103A-V104I-G118R-S128L-G159D-S188D- +A232V-Q245R-N248D-E271F G097P-S099A-S103G +A016S-T033S-S101G-S103A-V104I-N116L-S128N-Y209A-G211Q- + T213A-A232VS078G-G118S + T033S-G118R-S128I-A158E-S166D-S188D +S024R-N062E-S101A-S103A-V104I-S188D-M222S-A232V-Q245R +S099T-S101A-G102A-S103A-V104L + S056F +T022A-G097A-S101Q-S103A-V104I-L124V-L126I-S128A-P129E- +G159D-S188D-A232V-Q245R-N248D-E271F T022L-S024F-S128D-L217E +T022A-S101Q-S103A-V104I-L124V-L126I-S128A-G159D-S188D- +A232V-Q245R-N248D-E271F G020R-S078G-G118S-H249R +

TABLE 2-2 BMI cleaning performance of Group D2 variants. PI =Performance Index PI > or = 3 is +++; PI between 2.9 and 2 = ++; PIbetween 1.9 and 1.1 = + in Detergent Composition 104 as described inTable 1 PI Relative to GG36; BMI assay, Detergent Sequence of GG36Variants (BPN′ numbering) 104 at 25° C.A016S-S024F-S101D-S103A-V104I-N116A-S128N-Y209V-T213A- +++ A232VA016S-S024F-S101G-S103A-V104I-N116A-S128N-S130D-Y209V- +++ T213A-A232VA016S-S024F-S101G-S103A-V104I-N116A-S128N-S166D-Y209V- +++ T213A-A232VA016S-S024F-S101G-S103A-V104I-N116A-S128N-S188D-Y209V- +++ T213A-A232VA016S-S024F-T033S-S101G-S103A-V104I-N116L-Y209A-T213A- +++ A232VA016S-S024R-S101G-S103A-V104I-N116A-S128N-Y209V-T213A- +++ A232VA016S-S101G-S103A-V104I-N116L-S128N-Y209A-G211Q-A232V +++A016S-T022A-S024F-S101G-S103A-V104I-N116A-S128N-Y209V- +++ T213A-A232VA016S-T022A-S101G-S103A-V104I-N116A-Y209V-G211Q-T213A- +++ A232VA016S-T033S-S101G-S103A-V104I-N116A-Y209A-G211Q-A232V +++A098Q-G102A-S103G A098Q-S099T-G102A-S103G +++ G020K-N062E-N116L-T213A+++ G020K-N062E-N116L-T213A-M222S +++ G020K-N062E-S188D-T213A-M222S +++G020K-S024F-S166D-T213A-L217E +++ G020K-S166D-L217E +++G020K-T022L-G118R-P129E-S188D +++G020K-T022L-S024F-S078N-G118R-S166D-T213A-L217E +++G020K-T022L-S078N-G118R-S128D-T213A-L217E +++G020R-G061W-P129E-G159D-S188D-Q245R +++ G020R-G118R +++G020R-G118R-G159D-N248D-H249R +++ G020R-G118R-G159D-S188D-N248D +++G020R-G118R-G159D-S188D-Q245R-N248D-H249R +++ G020R-G118R-P129E +++G020R-G118R-P129E-G159D-H249R +++ G020R-G118R-P129E-GI59D-Q245R-N248D+++ G020R-G118R-P129E-G159D-Q245R-N248D-H249R +++G020R-G118R-P129E-G159D-S188D +++ G020R-G118R-P129E-S188D +++G020R-G118R-P129E-S188D-N248D +++ G020R-G118R-S188D-N248D +++G020R-G118R-S188D-Q245R-N248D +++ G020R-G118R-S188D-Q245R-N248D-H249R+++ G020R-G159D-N248D-H249R +++ G020R-G159D-Q245R-N248D +++G020R-G159D-S188D +++ G020R-G159D-S188D-H249R +++G020R-G159D-S188D-Q245R +++ G020R-G159D-S188D-Q245R-N248D +++G020R-N043R-S101A-P210I-G211Q +++ G020R-N062E-G118R +++G020R-N062E-G118R-G159D-N248D +++ G020R-N062E-G118R-S188D-N248D-H249R+++ G020R-N062E-S078G-G118S-G159D-S188D +++G020R-N062E-S078G-G118S-S188D-Q245R +++G020R-N062E-S078R-P129E-S188D-N248D +++ G020R-P129E +++G020R-P129E-G159D-Q245R-N248D +++ G020R-P129E-G159D-Q245R-N248D-H249R+++ G020R-P129E-G159D-S188D-Q245R +++ G020R-P129E-H249R +++G020R-P129E-N248D +++ G020R-P129E-Q245R +++ G020R-P129E-Q245R-N248D +++G020R-P129E-S188D-H249R +++ G020R-P129E-S188D-N248D-H249R +++G020R-P129E-S188D-Q245R +++ G020R-S024R-G118R-G159D-S188D-Q245R-N248D+++ G020R-S024R-G118R-P129E-G159D-N248D +++G020R-S024R-G118R-P129E-G159D-N248D-H249R +++G020R-S024R-G118R-P129E-G159D-S188D-N248D-H249R +++G020R-S024R-G118R-S188D-N248D +++ G020R-S024R-N062E-P129E-S188D-N248D+++ G020R-S024R-N062E-S078D-G118S-P129E-Q245R-N248D-H249R +++G020R-S024R-N062E-S078D-G118S-P129E-S188D-Q245R +++G020R-S024R-N062E-S078G-G118D-S188D-N248D +++G020R-S024R-N062E-S078G-G118S-G159D-N248D +++G020R-S024R-N062E-S078G-G118S-P129E +++G020R-S024R-N062E-S078G-G118S-Q245R +++ G020R-S024R-P129E-G159D +++G020R-S024R-P129E-G159D-N248D-H249R +++G020R-S024R-P129E-G159D-S188D-H249R +++G020R-S024R-P129E-G159D-S188D-N248D +++ G020R-S024R-P129E-H249R +++G020R-S024R-S078D-G118D-P129E-Q245R-H249R +++G020R-S024R-S078D-G118S-G159D-Q245R +++G020R-S024R-S078D-G118S-P129E-G159D-Q245R-H249R +++G020R-S024R-S078D-G118S-S188D-Q245R +++ G020R-S024R-S078G-G118S-G159D+++ G020R-S024R-S078G-G118S-G159D-Q245R-N248D +++G020R-S024R-S101A-P210I-G211Q +++ G020R-S024R-S188D +++G020R-S024R-S188D-Q245R-N248D +++G020R-S078G-G118S-P129E-G159D-S240E-N248D-H249R +++G020R-S078R-G118R-P129E-S188D-N248D +++G020R-S078R-G159D-S188D-Q245R-N248D +++G020R-S078R-P129E-G159D-S188D-H249R +++ G020R-S188D-H249R +++G020R-S188D-Q245R-N248D-H249R +++G061P-S078N-G097A-S101N-S128A-V203Y-L217Q +++ G097A-S101N-S128A-L217Q+++ G097P-A098Q-S099A-V1041 +++ G097P-A098Q-S099T-S101G +++G097P-S099A-G102A +++ G118R-P129E-G159D-Q245R-N248D +++G118R-P129E-G159D-S188D-H249R +++G118R-P129E-G159D-S188D-K237R-N248D-H249R +++ G118R-P129E-H249R +++G118R-S188D-N248D-H249R +++ G159D-S188D +++ G159D-S188D-Q245R-N248D +++N018K-G020R-S024R-R045T-S101A-P210I-G211Q-T213A +++N062E-G118R-P129E-S188D-Q245R +++ N062E-G159D-S188D-H249R +++N062E-P129E-G159D-S188D-Q245R +++ N062E-S078G-G118D-N248D-H249R +++N062E-S078R-G118R-G159D-S188D-P210L-N248D +++N062E-S078R-G118R-P129E-N248D +++ N062E-S078R-G159D +++N062E-S078R-G159D-N248D +++ N062E-S078R-P129E-G159D +++N062E-S078R-P129E-S188D-Q245R-N248D +++ N062E-S078R-Q245R-N248D +++P086S-S087G-A088V-G097A-N117S-S128A-L217Q +++ P129E-G159D +++P129E-G159D-Q245R-N248D +++ P129E-G159D-S188D-H249R +++P129E-Q245R-N248D-H249R +++ S024F-G118R-S128D +++S024F-S078N-T213A-L217E +++S024G-G061S-S078N-S101N-Q109G-S128A-L217Q-N243V +++S024G-S078N-A088T-S101N-Q109G-N116T-S128A-A158S-L217Q- +++ N218S-L257GS024G-S078N-S101N-Q109G-L217Q-N243V-N248A +++S024G-S078N-S101N-Q109G-N116T-S128A-L217Q-N243V-S256R +++S024G-S078N-S101N-Q109G-S128A-L217Q +++S024G-S078N-S101N-Q109G-S128A-L217Q-S256R +++S024G-S078N-S101N-S128A-A158S-L217Q-L257G +++S024G-S078N-S101N-S128A-L217Q +++ S024G-S078N-S101N-S128A-L217Q-N243V+++ S024R-G118R-G159D-S188D +++ S024R-G118R-G159D-S188D-N248D +++S024R-G118R-P129E-G159D-S188D-Q245R-N248D +++S024R-G118R-P129E-G159D-S188D-Q245R-N248D-H249R +++S024R-G118R-P129E-S188D-H249R +++ S024R-G118R-S188D +++S024R-G118R-S188D-N248D +++ S024R-G159D-S188D-Q245R +++S024R-N062E-G159D-S188D-Q245R +++ S024R-N062E-N116L-G118R-S128I-A158E+++ S024R-N062E-N116L-G118R-S128I-S166D +++ S024R-N062E-Q245R +++S024R-N062E-S078D-G118S-S188D-Q245R +++S024R-N062E-S078G-G118D-N248D-H249R +++S024R-N062E-S078G-G118S-S188D-Q245R-N248D +++S024R-N062E-S078R-G159D-N248D +++ S024R-N062E-S078R-P129E-H249R +++S024R-N062E-S188D-N248D +++ S024R-P129E-G159D-Q245R-N248D +++S024R-P129E-G159D-S188D-Q245R-N248D-H249R +++ S024R-P129E-Q245R-N248D+++ S024R-S078R-G118R-P129E-G159D-S188D +++ S024R-S078R-G159D-S188D +++S024R-S078R-G159D-S188D-Q245R-N248D-H249R +++ S024R-S188D-N248D +++S024R-S188D-Q245R-N248D +++ S078N-G118R-S166D-L217E-M222S +++S078R-G118R-P129E +++ S078R-G118R-P129E-S188D-N248D +++S078R-L111I-N185I-I198L-L217E +++ S078R-P129E-G159D +++S078R-V084A-P129E-Q245R +++ S099T-S101A-G102A-S103A +++S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F +++S101G-S103A-V104I-N116L-G159D-S188D-A232V-Q245R-N248D- +++ E271FS188D-Q245R-N248D +++T022A-G097A-S101G-S103A-V104I-L124V-P129S-G159D-S188D- +++A232V-Q245R-N248D-E271FT022A-G097A-S101N-S103A-V104I-S128A-G159D-S188D-A232V- +++Q245R-N248D-E271F T022A-G097A-S101N-S103A-V104I-S128A-G159D-S188D-L217Q-+++ A232V-Q245R-N248D-E271FT022A-G097A-S101Q-S103A-V104I-L124V-P129S-G159D-S188D- +++A232V-Q245R-N248D-E271F T022A-G097A-S101Q-S103A-V104I-P129S-A232V-Q245R+++ T022A-G097S-S101N-S103A-V104I-S128A-G159D-S188D-L217Q- +++A232V-Q245R-N248D-E271FT022A-G097S-S101N-S103A-V104I-S128A-G159D-S188D-L217Q- +++M222S-A232V-Q245R-N248D-E271FT022A-S024F-S101G-S103A-V104I-N116L-S128N-Y209A-G211Q- +++ T213A-A232VT022A-S024G-S078N-S101N-S103A-V104I-G159D-S188D-L217Q- +++A232V-Q245R-N248D-E271F T022A-S024R-N062E-N116L-G118R-A158E +++T022A-S024R-T033S-N062E-N116L-G118R-P129E-S188D +++T022A-S024R-T033S-N116L-A158E-S166D-S188D +++T022A-S101G-S103A-G159D-S188D-A232V-Q245R-N248D-E271F +++T022A-S101G-S103A-N116L-G159D-S188D-A232V-Q245R-N248D- +++ E271FT022A-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-E271F +++T022A-S101G-S103A-V104I-G159D-S188D-A232V-N248D-E271F +++T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-E271F +++T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D +++T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D- +++ E271FT022A-S101G-S103A-V104I-G159D-S188D-M222S-A232V-Q245R- +++N248D-A270V-E271F T022A-S101G-S103A-V104I-G159D-S188D-M222S-A232V-Q245R-+++ N248D-E271F T022A-S101G-S103A-V104I-G159D-S188D-Q245R-N248D-E271F+++ T022A-S101G-S103A-V104I-G159D-S188E-A232V-Q245R-N248D +++T022A-S101G-S103A-V104I-L217Q-A232V-Q245R +++T022A-S101G-S103A-V104I-M119V-P129E-G159D-S188D-L217Q- +++A232V-Q245R-N248D-E271FT022A-S101G-S103A-V104I-N116L-G159D-A232V-Q245R-N248D- +++ E271FT022A-S101G-S103A-V104I-N116L-G159D-S188D-A232V-N248D- +++ E271FT022A-S101G-S103A-V104I-N116L-G159D-S188D-A232V-Q245R- +++ E271FT022A-S101G-S103A-V104I-N116L-G159D-S188D-A232V-Q245R- +++ N248D-E271FT022A-S101G-S103A-V104I-N116L-G159D-S188D-Q245R-N248D- +++ E271FT022A-S101G-S103A-V104I-N116L-S128N-Y209V-G211Q-T213A- +++ A232VT022A-S101G-S103A-V104I-N116L-S188D-A232V-Q245R-N248D- +++ E271FT022A-S101G-S103A-V104I-N116L-Y209V-G211Q-A232V +++T022A-S101G-S103A-V104I-P129S-L217Q-A232V-Q245R +++T022A-S101G-S103A-V104I-S128A-P129S-L217Q-A232V-Q245R +++T022A-S101G-S103A-V104I-S128L-G159D-S188D-T213A-A232V- +++Q245R-N248D-E271F T022A-S101G-S103A-V104I-S188D-A232V-Q245R-N248D-E271F+++ T022A-S101G-V104I-G159D-S188D-A232V-Q245R-N248D-E271F +++T022A-S101G-V104I-N116L-G159D-S188D-A232V-Q245R-N248D- +++ E271FT022A-S101Q-S103A-V104I-L124V-S128A-P129S-A232V-Q245R +++T022A-S101Q-S103A-V104I-L126I-P129S-G159D-S188D-L217Q- +++A232V-Q245R-N248D-E271F T022A-S101Q-S103A-V104I-P129S-L217Q-A232V-Q245R+++ T022A-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F +++T022A-S103A-V104I-N116L-G159D-S188D-A232V-Q245R-N248D- +++ E271FT022A-S101Q-S103A-V104I-L124V-S128A-P129S-A232V-Q245R +++T022A-S101Q-S103A-V104I-L126I-P129S-G159D-S188D-L217Q- +++A232V-Q245R-N248D-E271F T022A-S101Q-S103A-V104I-P129S-L217Q-A232V-Q245R+++ T022A-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F +++T022A-S103A-V104I-N116L-G159D-S188D-A232V-Q245R-N248D- +++ E271FT022A-T033S-S101G-S103A-V104I-N116L-Y209A-G211Q-T213A- +++ A232VT022L-S078N-G118R-S128D-L217E +++T022Q-S101G-S103A-V104I-G159D-S188D-A232L-Q245R-N248E- +++ E271HT022R-S101G-S103A-V104I-A232V-Q245R +++ A098F-S099A-S101G-V104I ++A098Q-S099G-S101G-V104L ++ A098Q-S099T-S101G-S103A ++ G020K-T022L-Q245R++ G020R-G118R-G159D-Q245R ++ G020R-P129E-G159D-N248D ++G020R-S024R-G118R-P129E-G159D-H249R ++ G020R-S024R-G159D-N248D ++G020R-S024R-S078D-G118S-Q245R ++G020R-S024R-S078G-G118S-Q245R-N248D-H249R ++G020R-S078D-G118S-P129E-Q245R-H249R ++ G020R-S078D-G118S-Q245R ++G020R-S078G-G118S-Q245R-N248D-H249R ++ G020R-S078R-G159D-S188D ++G020R-T022W-S024R-N043R-R045T-S101A-G211Q ++ G097A-S128A-L217Q ++ G100S++ G118R-H249R ++ G118R-P129E-N248D ++ P129E-S188D ++P129E-S188D-Q245R-H249R ++ S024G-N076D-S078N-S101N-H120Q-S128A-L217Q ++S024G-S078N-S101N-L217Q ++ S024G-S078N-S101N-S128A-L217Q-S256R ++S024R-G118R-N248D-H249R ++ S024R-P129E-G159D-N248D ++S024R-S078D-G118S-G159D-Q245R-H249R ++ S024R-S078R-G118R-G159D ++S078G-G118S-Q245R-H249R ++ S078G-G118S-S188D-Q245R-H249R ++S078R-G118R-G159D ++ S078R-G118R-G159D-N248D ++ S078R-P129E-N248D ++S078R-P129E-Q245R ++ S099T-G102A ++ S099T-S101G-G102A-S103A ++ S188D ++S188D-N248D ++ T022A-S101G-S103A-V104I-N116L-G159D-S188D-A232V-Q245R- ++N248D T022A-S101Q-S103A-V104I-P129S-A232V-Q245R ++T022K-S101A-S103N-V104L-A232T-Q245R ++T022W-N043R-R045T-S101A-P210I-T213A ++ A098F-S099A + A098F-S099A-V104I +A098Q-S099A-G100S + A098Q-V104L + G020R-N248D + G020R-S024R-Q245R +G020R-S024R-S078D-G118S-H249R + G020R-S078D-G118S-Q245R-H249R +G061E-G097A-S128A-P129E-G159K-L217Q + G097P-S099G-G100S +N062E-P129E-G159D-N248D + P129E-G159D-N248D + P129E-G159D-S188D +Q245R + Q245R-N248D-H249R + S024G-N076D-S078N-S101N-S128A-L217Q-N218S +S024R + S024R-S078G-G118S-Q245R + S099T-G100S-S103A-V104I +S099T-S101A-S103A + S099T-S103A-V104I + V104I +

TABLE 2-3 BMI cleaning performance of Group D3 variants. PI =Performance Index PI > or = 3 is +++; PI between 2.9 and 2 = ++; PIbetween 1.9 and 1.1 = + in Detergent Composition 105 as described inTable 1 PI Relative to GG36; BMI assay, Detergent Sequence of GG36Variants (BPN′ numbering) 105, 25° C. G020K-S101D-G102A-L217E-S240R +++N043R-G102A-L217E +++ N062E-S078N-S101D-S240R +++ G102A-L217E +++G020K-S128L-L217E-S240R +++ G020K-N062E-S078N-L217E-S240R +++N043R-S101D-S128L-L217E-S240R +++ G020K-S078N-S101D-G102A-P210L +++N043R-S101D-L217E +++ G020K-G100S-L217E-S240R +++ S078N-G100S +++G020K-N043R-S101D-G102A-L217E +++ G100S-S240R +++ G020K-G102A-L217E +++G020K-S024G-N062E-S078N-S101N-Q109N-N116L-S128A-S188D +++G020K-L217E-S240R +++ N043R-S101D +++ N043R-S078N-S101D-S128L-S240R +++N043R-S078N-S101D-L217E-S240R +++ G020K-S078N-S101D +++N043R-S128L-S240R +++ G020K-N062E-N116L-N123G-T213A +++G020K-N062E-G097S-S101G-Q109G-N116L-S128A +++G020K-N062E-S078N-G097A-S101N-Q109N-N116L-S188D +++N043R-S078N-S101D-G102A-L217E +++ G020K-S078N-S099G-S128L-L217E +++G020K-N062E-S101D +++ G020K-S128L-L217E +++G020K-S024G-N062E-S078N-G097S-S101G-Q109G-N116L-L217Q +++N043R-G097A-S101D-S128L +++ G020K-S024G-N062E-S101Q-Q109N-N116L-S128A+++ G020K-S024G-N062E-S078N-S101N-N116L-S128A-L217Q +++S078N-G102A-S240R +++ N043R-S078N-G100S-L217E-S240R +++G020K-S078N-S128L-L217E-S240R +++ G020K-N062E-N116L-S128Q +++G020K-S101G-L217E-S240R +++G020K-S024G-N062E-S101N-Q109G-N116L-S128A-L217Q +++G020K-N062E-S078N-G097S-S101N-N116L-L217Q +++G020K-N062E-G097S-S101N-Q109G-N116L-L217Q +++G020K-S024G-N062E-S078N-G097A-S101Q-Q109N-N116L-M222S +++G020K-S024G-N062E-S078N-G097S-S101Q-Q109N-N116L-S128A- +++ S188D-L217QG020K-N062E-S078N-S101N-Q109G-N116L-S188D-L217Q +++G020K-N062E-S105G-N116L-S128L-T213A +++G020K-N062E-G097A-S101Q-Q109G-N116L-S188D-L217Q ++G020K-N062E-S078N-G097A-S101Q-Q109G-N116L ++G020K-N062E-G097A-S101Q-Q109N-N116L-L217Q ++N043R-S078N-G102A-S128L-L217E-S240R ++ S078N-G097S-S101Q ++N043R-N062E-S078N-S101D-S128L-S240R ++ S128L-L217E-E271L ++G020K-S024G-N062E-G097S-S101N-Q109N-N116L-S128A-L217Q ++G020K-N062E-N116L-S128L-T213A ++ S078N-G097A-S101G-Q109G-S188D-L217Q ++S101D-S128L-L217E-S240R ++G020K-S024G-N062E-G097A-S101Q-Q109G-N116L-S128A ++G020K-N062E-S078N-G097S-S101N-Q109N-N116L-S128A-S188D- ++ L217QG020K-N062E-G097S-S101Q-Q109G-N116L-S128A-S188D-L217Q ++G020K-S024G-N062E-S078N-G097S-S101N-Q109N-N116L-S128A ++S024G-S078N-G097S-S101G-S128A-S188D-L217Q ++S024G-S078N-G097A-S101N-Q109G-S128A-S188D-L217Q ++G020K-N043R-G102A-S128L ++ G020K-S078N-S101D-G102A-L217E-S240R ++G020K-N062E-G100S ++ S078N-S128L-L217E-E271L ++ S128Q-L217E-E271L ++G020K-L217E ++ G020K-S024G-N062E-S078N-G097A-S101N-N116L-S128A-L217Q ++G020K-S024G-N062E-S078N-G097S-S101G-N116L-S128A-L217Q ++G020K-S078N-S101D-G102A-L217E ++ G020K-N062E-S128L ++G020K-N062E-S078N-S101G-Q109N-N116L ++G020K-S024G-N062E-S078N-G097S-S101Q-N116L-S128A-L217Q ++S078N-S128L-L217E-S240R ++ S101D-S128Q-L217E-S240R-E271L ++N043R-G102A-S240R ++ G020K-N062E-S078N-S101N-Q109N-N116L-M222S ++G020K-N062E-S101N-Q109G-N116L-S128A-L217Q ++G020K-S024G-N062E-S078N-G097A-S101G-N116L-L217Q ++G020K-N062E-S101G-N116L-S188D ++G020K-N062E-G097S-S101Q-Q109N-N116L-S128A-S188D ++G020K-I035V-G100S-S128L-L217E ++ N043R-S101D-G102A-S240R ++N062E-S078N-G100S ++ G020K-N043R-N062E-A215T ++ N062E-S240R ++N043R-S128L-L217E-S240R ++ N062E-S101D-S240R ++ G020K-S101D-N116L-T213A++ G020K-S101D-N116L-S128Q-P210S-T213A ++ G020K-S101D-T213A ++G020K-S024G-N062E-S078N-G097S-S101Q-Q109N-N116L-S128A ++G020K-N062E-S078N-G097S-S101Q-N116L-S128A-L217Q ++T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D- ++ E271FS024G-G097S-S101N-Q109G-S188D-L217Q ++G020K-S024G-N062E-S101N-Q109G-N116L-L217Q ++ L217E-S240R ++S101D-L217E-S240R-E271L ++ N043R-N062E-S101D-S128L-S240R ++G097A-S101N-S128A-S188D-L217Q ++G020K-N062E-S078N-S101N-N116L-S128A-L217Q ++G020K-N062E-S078N-G097S-S101G-Q109N-N116L-L217Q ++G020K-S024G-N062E-G097S-S101N-Q109G-N116L-S128A-L217Q ++G020K-S024G-N062E-G097S-S101G-Q109G-N116L-S128A-S188D- ++ L217QG020K-N062E-S101N-Q109N-N116L-S128A-S188D ++S078N-G097S-S101N-Q109N-S128A-S188D-L217Q-W241L ++G020K-N043R-N062E-S078N-S101D ++ G020K-N062E-S101D-S240R ++G020K-S101D-S128Q ++ G020K-N116L-S128Q-Y209H-T213A ++G020K-N043R-S078N-G100S-L217E-N218D ++S024G-S101N-Q109G-S128A-S188D-L217Q ++G020K-S024G-N062E-S078N-G097S-S101N-Q109G-N116L-S128A- ++ S188DS078N-S101N-Q109G-S188D-L217Q ++G020K-S024G-N062E-S078N-G097S-S101N-Q109G-N116L-S188D- ++ L217QG020K-N062E-S078N-S101Q-Q109N-N116L-S128A-S188D-L217Q ++G020K-N062E-S101G-Q109G-N116L ++G020K-S024G-N062E-G097S-S101N-Q109G-N116L-S128A-S188D- ++ M222SS024G-G097A-S101G-S188D-L217Q ++ S024G-S078N-G097A-S101G-S128A-S188D ++G097S-S101N-Q109G-S128A-S188D ++ G020K-S078N-S101D-I246T ++N043R-S128L-L217E ++ G020K-S101D-L217E-S240R ++ S128L-L217E-T224A-E271L++ G020K-S078N-S101D-S128Q-L217E-E271L ++ N062E-S078N-S101D-L217E-S240R++ G020K-S101D-L217E-E271L ++ G020K-S078N-S128Q-L217E-S240R ++G020K-N062E-S101D-N116L-T213A ++ G100S-S101D ++S078N-G097S-S101G-S128A-L217Q ++G020K-N062E-G097S-S101N-N116L-S128A-L217Q ++G020K-N062E-G097S-S101N-N116L-L217Q ++ S101N-Q109G-S188D ++G020K-S024G-N062E-G097S-S101Q-Q109N-N116L-S128A-L217Q ++G020K-S024G-N062E-G097S-S101N-N116L ++G020K-S024G-N062E-S101N-N116L-S128A ++G020K-S024G-N062E-S078N-S101Q-Q109N-N116L-S128A ++G020K-N062E-S078N-S101N-Q109G-N116L-S188D ++G020K-N062E-S078N-S101N-Q109G-N116L-S128A ++G020K-S024G-N062E-S078N-G097S-S101N-Q109G-N116L-S128A- ++ M222SG020K-S024G-N062E-S078N-G097S-S101G-N116L-S128A ++S024G-S078N-G097S-S101Q-Q109G-S128A-S188D-L217Q ++S024G-S078N-S101G-Q109N-S188D-L217Q ++S024G-S078N-G097A-S101G-Q109G-S188D ++S078N-S101D-S128Q-L217E-S240R-E271L ++ G020K-S128Q-L217E ++S128Q-L217E-S240R-E271L ++ G020K-S078N-S101D-L217E ++G020K-S078N-S101D-L217E-E271L ++ G020K-N116L-S128Q-T213A ++G020K-N116L-N123G ++ S101G-S188D-L217Q ++ L217E ++G020K-S024G-N062E-S078N-S101G-N116L-L217Q ++S078N-G097A-S101G-Q109N-S128A-S188D ++ G020K-S078N-S128L-L217E-E271L ++G020K-N062E-S101Q-Q109G-N116L ++ G020K-S101D-L217E ++S024G-G097A-S101N-Q109N-S128A ++ G020K-S078N-S101D-S128L-L217E-S240R ++G097A-S101G-L217Q ++ S024G-S101Q-Q109G-S128A-L217Q ++G020K-N062E-N116L-T213A-M222S ++G020K-S024G-N062E-T071A-S078N-G097S-S101G-N116L-L217Q ++G020K-S024G-N062E-S078N-S101G-N116L-S128A ++G020K-S024G-N062E-S078N-G097A-S101N-N116L ++G020K-N062E-G097A-S101N-N116L ++G020K-S024G-N062E-G097A-S101N-Q109G-N116L-S128A ++G020K-S024G-N062E-S078N-G097S-S101G-Q109N-N116L ++G020K-N062E-S078N-G097S-S101Q-Q109G-N116L-S128A-S188D- ++ L217QG020K-N062E-S101N-Q109G-N116L-L217Q ++G020K-S024G-N062E-G097A-S101N-Q109G-N116L-S188D-L217Q ++G020K-S024G-N062E-S101G-N116L ++ S078N-G097S-S101N-Q109G-G159D-L217Q ++S024G-G097A-S101N-Q109G-S188D ++ S078N-S101G-S128A-L217Q ++S078N-G097A-S101N-S128A-S188D-L217Q ++S024G-G097S-S101G-S128A-S188D-L217Q ++S024G-S078N-G097S-S101Q-Q109G-S128A-S188D-L217Q-V227A ++G020K-N062E-S078N ++ S078N-S128L-L217E-S240R-E271L ++G020K-N062E-I072N-S101D-N116L-S128Q ++G020K-T057P-N062E-N116L-S128Q-T213A ++ G020K-N043R-S078N-S101D-S240R ++S078N-G097S-S101N-Q109N-S128A-S188D-L217Q ++S024G-S101N-Q109N-S128A-S188D ++G020K-S024G-N062E-G097S-S101Q-N116L-S128A ++S024G-S078N-G097S-S101N-Q109N-S128A-S188D ++ N043R-G100S ++G020K-N062E-S078N-S101Q-Q109G-N116L-S128A ++S078N-S101Q-Q109N-S188D-L217Q ++ G020K-N116L-S128L-T213A ++G020K-S078N-S101D-L217E-S240R ++ G020K-N062E-S101N-Q109N-N116L-M222S ++G020K-S024G-N062E-G097A-S101Q-Q109N-N116L-S128A ++S078N-G097S-S101G-S128A-S188D ++ G097S-S101G-Q109N-S128A-M222S ++S024G-S078N-G097A-S101Q-S128A-M222S ++ G097A-S101G-Q109N-L217Q ++S024G-S078N-S101G-S128A-S188D-L217Q ++S024G-G097A-S101N-Q109G-S128A-S188D ++ G097S-S101N-Q109G-S128A ++S078N-G097A-S101Q-L217Q ++ G020K-N043R-S078N-S101D ++G020K-N043R-E054D-N062E-S078N-S240R ++ G020K-S101D-S240R ++S128Q-L217E-S240R ++ G020K-S128Q-L217E-V244A-E271L ++ G020K-L217E-E271L++ G020K-S078N-S128L-L217E ++ S078N-S101D-S128Q-L217E-E271L ++G020K-N062E-L217E-E271L ++ G020K-N062E-S101D-N116L-S128Q-T213A ++G020K-N062E-S078N-N116L-S128I ++ G020K-N062E-S078N-S128Q-T213A ++S024G-G097A-S101G-Q109G-S128A ++ S024G-G097S-S101N-Q109N-S128A-L217Q ++G020K-S024G-N062E-S078N-S101N-N116L-S128A-S188D-L217Q ++ G020K-S101D ++G020K-N062E-S078N-G097S-S101Q-N116L-S128A ++G020K-N062E-G097S-S101G-N116L-S188D ++ N043R-S078N-G100S-S128L-S240R ++G097S-S101N-Q109N-S188D ++ G097S-S101Q-Q109N-S128A-L217Q ++G020K-N062E-S101N-Q109G-N116L-S128A-S188D-M222S ++G020K-S024G-N062E-S078N-G097S-S101Q-Q109G-N116L ++ G020K-N062E-M222S ++G097S-S101Q-Q109G-S188D ++ G020K-N062E-N116L-T213A ++G020K-S078N-L217E-S240R ++G020K-N062E-S078N-G097A-S101N-Q109G-N116L-S188D ++G020K-S024G-N062E-G097S-S101Q-Q109G-N116L-S188D ++G020K-N062E-S078N-G097A-S101Q-N116L-S128A-M222S ++G020K-N062E-S078N-S101N-N116L-S188D ++G020K-S024G-N062E-S078N-S101G-N116L ++G020K-N062E-S078N-S101N-N116L-S128A-S188D ++G020K-S024G-N062E-G097S-S101N-Q109G-N116L-S128A-S188D- ++ L217QG020K-N062E-G097S-S101N-Q109N-N116L ++G020K-N062E-S101G-N116L-S188D-L217Q ++ G097A-S101N-S128A ++S101G-Q109N-S128A-M222S ++ S024G-G097S-S101Q-Q109G-S188D-L217Q ++G097S-S101Q-Q109N-S128A-S188D ++ S024G-S078N-G097A-S101Q-S188D-L217Q ++S024G-S078N-S101G-Q109G-S128A-L217Q ++ G020K-N062E-S128L-S240R ++N062E-S078N-S128L-S240R ++ N043R-G102A-S128L-L217E-S240R ++N062E-S078N-L217E-S240R-E271L ++ S078N-S128Q-L217E-N269S ++G020K-N062E-N123G-T213A ++G020K-N062E-S078N-S099G-N116L-S128Q-T213A-L217N ++G020K-N062E-S078N-S128I-L217N ++ G097A-S101Q-S128A-S188D-L217Q ++G020K-N062E-N116L ++ G020K-N062E-S078N-G097S-S101N-Q109G-N116L ++G020K-S024G-N062E-S078N-G097S-S101N-Q109G-N116L-S188D ++G020K-N043R-S128L-S240R ++ S024G-S078N-G097A-S101N-Q109N-S128A ++G020K-N062E-G097S-S101Q-N116L-L217Q ++ G020K-N062E-S078N-S128I ++G020K-S024G-N062E-L090V-G097A-S101Q-N116L-M222S ++G020K-N062E-S078N-S101Q-Q109G-N116L-S188D-L217Q ++G020K-S024G-N062E-G097A-S101G-N116L-S128A-S188D-L217Q ++G020K-N062E-G097S-S101N-Q109N-N116L-S188D-L217Q ++G020K-S024G-A048V-N062E-S078N-G097A-S101N-N116L-S128A- ++ L217QG020K-N062E-S078N-S101Q-Q109G-N116L ++G020K-S024G-N062E-G097S-S101N-Q109N-N116L-S128A ++G020K-N062E-S078N-G097A-S101Q-Q109G-N116L-S188D ++G020K-N062E-S078N-G097A-S101N-Q109N-N116L-N218D ++S024G-S101N-Q109G-S128A-S188D-M222S ++S024G-G097A-S101Q-Q109N-S188D-L217Q ++S024G-S078N-G097S-S101Q-Q109N-S128A-S188D ++ S101N-Q109G-M222S ++S024G-I072V-G097S-S101N-S128A-L217Q ++ S101G-S128A ++N043R-S078N-G102A-A215G ++ G020K-S101D-S128L-L217E-S240R ++S078N-S101D-S128Q-L217E-S240R ++ G020K-N116L-S128L-M222S ++G020K-N062E-S078N-N116L-T213A-M222S ++ G020K-N062E-S078N-N116L-S128Q ++G020K-N062E-T213A ++ G020K-N062E-S078N-N116L-N123G-M222S ++S024G-S078N-G097A-S101N-Q109N-L217Q + G020K-G102A-S128L +G020K-S078N-S101D-S128Q + S024G-S078N-G097A-S101N-S128A +G020K-S024G-N062E-S101Q-Q109G-N116L-S188D + G020K-S078N-S101D-S240R +G020K-N043R-S128L-L217E-S240R + S078N-S101G-Q109N-L217Q +S024G-G097S-S101N-S128A-S188D + S078N-L217E-E271L +G097S-S101Q-Q109N-L217Q + G020K-N062E-N116L-M222S +G020K-N062E-S078N-N116L-S128Q-T213A +G020K-S024G-N062E-S101Q-Q109G-N116L-S188D-L217Q +S024G-S078N-G097A-S101Q-S128A-L217Q + S078N-S101D-S128L-E271L +G020K-N062E + G020K-N062E-S101N-N116L +G020K-N062E-S101Q-Q109N-N116L-M222S +G020K-N062E-S101Q-Q109G-N116L-M222S +G020K-S024G-N062E-S078N-G097S-S101G-Q109G-N116L-L217Q- + M222SG020K-S024G-N062E-S078N-S101N-N116L-S128A-M222S +G020K-N062E-S078N-S101N-Q109N-N116L-S188D-M222S +G020K-S024G-N062E-S101N-Q109N-N116L-S188D-M222S +G020K-S024G-N062E-G097S-S101Q-N116L-L217Q +G020K-N062E-G097S-S101N-Q109G-N116L-S128A-S188D +S078N-G097A-S101Q-Q109G-S128A + S024G-G097A-S101N +S024G-G097A-S101Q-Q109N-S128A-S188D + G097A-S101N-S128A-S188D +S024G-G097S-S101G-S128A-L217Q + S078N-G097A-S101G-Q109N-S128A-L217Q +S024G-G097S-S101Q-Q109N-S128A + G097A-S101N-Q109G-S128A +S024G-G097A-S101Q-Q109G-L217Q +S024G-S078N-A085G-G097A-S101N-S128A-S188D-M222S +S024G-S078N-G097S-S101Q-L217Q + S024G-S101D-S128Q-L217E +S078N-G097A-S101N-M222S +S024G-N076K-S078N-G097A-S101G-S128A-S188D-M222S +S024G-S101G-S128A-M222S + S024G-S078N-G097A-S101G-S188D +G102A-L217E-N218D + S078N-G100S-S101D +G020K-N043R-N062E-S078N-S128L-L217E + G020K-N043R-S101D-S128L +G020K-G100S-S101D-S128L-S240R + S078N-S128Q-L217E +G020K-S078N-L217E-E271L + N062E-S078N-L217E + N062E-L217E +G020K-S078N-S101D-S128Q-L217E + G020K-S078N-S101D-S128L-S240R + S128L +G020K-N123G + G020K-S128Q-T213A-M222S + S078N-L217E +G020K-N062E-S128I-L217N + G020K-N062E-S078N-S128Q-L217N +G097S-S101N-Q109G-S128A-S188D-M222S +G020K-S024G-N062E-S101Q-Q109N-N116L-S188D +G020K-N062E-S078N-N116L-S128I-L217N +S024G-S078N-G097A-S101N-Q109G-S188D + S024G-G097A-S101Q-Q109N-L217Q +G020K-S101D-S128L + S101N-Q109G-S128A + S078N-S101D-L217E +S024G-S078N-S101N-S128A-M222S +G020K-S024G-N062E-G097A-S101Q-N116L-S128A-S188D +S024G-S078N-G097A-S101N-Q109N-S128A-M222S +G020K-N062E-S078N-G097A-S101N-N116L-S128A-S188D-L217Q +G020K-S024G-N062E-S078N-G097S-S101Q-N116L-S188D +G020K-N062E-S078N-G097S-S101G-N116L-M222S +G020K-S024G-N062E-S078N-S101N-N116L-M222S +G020K-S024G-N062E-G097S-S101Q-N116L +G020K-S024G-N062E-S101Q-Q109G-N116L-S128A-S188D +G020K-S024G-N062E-G097S-S101N-Q109N-N116L-S128A-S188D- + L217QG020K-N062E-S078N-G097S-S101N-N116L + S024G-S078N-S101G-Q109G-M222S +S078N-G097A-S101G-Q109G-S128A + S078N-G097S-S101N-Q109N-S128A +S078N-G097A-S101N-Q109N-S188D + S024G-S101N-S128A-S188D-M222S +S101Q-Q109N-S188D-M222S + S101D-G102A-L217E +G020K-N062E-S078N-S101D-L217E + G020K-S078N-S101D-I107T-S128L-S240R +G020K-S078N-G100S-L217E-N218D + G020K-N043R-S101D +N043R-G100S-S128L-L217E + G020K-S078N-S101D-S128L-L217E-E271L +G020K-S101D-S128L-E271L + G020K-S101D-N116L-S128L-T213A +G020K-N062E-S078N-T213A-L217N + G020K-N062E-N116L-L217N +G020K-N062E-S128I-T213A + N062E-S101D-S128L-S240R + G097S-S101N-S128A +G020K-N062E-S078N-N116L-T213A-L217N + G020K-S101D-M222S +S024G-S101G-Q109G + G097A-S101Q-Q109G + G097S-S101Q-Q109N-S188D +G020K-N062E-G097S-S101N-N116L-S128A-S188D-L217Q +S078N-S101Q-S128A-M222S + S078N-G097A-S101Q-Q109N +S024G-S078N-G097A-S101Q-S128A-S188D + S078N-S101D-G102A +G020K-N062E-S078N-G097S-S101Q-Q109G-N116L-S128A-S188D- + M222SG020K-S024G-N062E-S078N-G097S-S101N-Q109N-N116L-S188D- + L217Q-M222SG020K-N062E-G097S-S101Q-N116L-S128A +G020K-S024G-N062E-G097S-S101Q-N116L-S128A-S188D-L217Q +S024G-S101N-Q109N-S128A + S024G-G097S-S101Q-S128A-S188D +S024G-S101Q-L217Q + G097S-S101G-S128A + S024G-S078N-S101Q-S128A-L217Q +S024G-S078N-G097S-S101N-S188D + S024G-G097A-S101N-S128A-M222S +G097A-S101Q-Q109N-S188D-M222S + S024G-S078N-G097S-S101Q +G020K-S101D-S128L-S212P-L217E-S240R +G020K-S101D-S128L-L217E-N218S-S240R + S101D-S128Q +G020K-N062E-S078N-E271L + S101D-E271L + S101D-S240R-E271L +G020K-S101D-E271L + G020K-N116L-S128L-T213A-M222S +G020K-N116L-S128Q-M222S + G020K-N062E-V084A-S128Q-M222S +G020K-N062E-S078N-N116L + S078N-S101N-Q109G-S128A-M222S +S024G-G097S-S101Q-Q109G + G020K-N062E-T213A-L217N +G097S-S101G-S128A-S188D + G020K-S240R + N062E-S078N +G020K-S128Q-M222S + S078N-G097S-S101G-Q109G +G020R-S024G-N043R-S078N-S101Q + G097S-S101N-Q109G-S128A-M222S +S024G-G097S-S101G-Q109N + S024G-S078N-G097A-S101G-Q109N +G020K-N062E-S078N-S101N-N116L-S128A-S188D-L217Q +G020K-N062E-S101Q-Q109G-N116L-S128A-M222S +G020K-S024G-N062E-S101Q-Q109N-N116L + S024G-S078N-S101Q-M222S +S024G-G097A-S101Q-Q109G-S128A + S101Q-Q109N-L217Q +S101Q-Q109G-S188D-L217Q + S024G-G097A-S101N-S188D-M222S +S024G-S078N-G097A-S101Q-Q109N + G097A-S101Q-S128A +G097A-S101Q-Q109G-S128A + S024G-G097S-S101Q-M222S +S078N-S101D-L217E-S240R + G020K-G100S-S128L +G020K-N043R-S078N-S128L-S240R + G020K-N043R-S101D-S240R +G020K-N043R-S078N-S128L + N062E-S128L + G020K-S078N-S101D-S128L +S078N-S101D + S101D + G020K-S101D-N116L-T213A-M222S +G020K-T213A-M222S + G020K-N116L-T213A + G020K-N116L +G020K-N062E-S128Q-L217N + S078N-G097S-S101G-Q109N-S128A +S078N-S101Q-Q109G + N062E-S078N-S128L +G020K-N062E-N116L-S128Q-T213A-L217N + G020K-N116L-T213A-M222S +G020K-S024G-N062E-S101Q-Q109H-N116L-M222S +G020K-N116L-S128Q-T213A-M222S + S024G-G097S-S101N +G020K-P055T-N062E-N116L-L217N + S024G-S101Q-S128A-S188D-M222S +G020K-M222S + G020K-N062E-S078N-G097A-S101N-N116L-S188D-L217Q +G097S-S101G-Q109N-M222S +G020K-S024G-N062E-S078N-G097A-S101N-N116L-S128A-L217Q- + M222SG020K-S024G-N062E-G097S-S101N-Q109N-N116L-S128A-S188D- + L217Q-M222SG020K-N062E-S078N-S101Q-N116L-S128A +S024G-S078N-S101N-Q109G-S188D-L217Q-M222S + S101N-Q109N-S128A-M222S +S101Q-Q109G-S128A-S188D + G097A-S101G-S188D-M222S +S078N-G097S-S101N-Q109G-S128A-S188D-L217Q-M222S +S024G-G097A-S101Q-M222S + S024G-G097S-S101N-Q109N-S128A-M222S +S024G-S078N-G097S-S101N-M222S + G020K-G102A-S240R + S078N-S128Q-E271L +G020K-N062E-S078N-S128Q + G020K-N062E-S078N-T213A-M222S +G020K-N062E-S078N-S128I-T213A-L217N + S078N-G097A-S101N +G020K-N062E-N116L-S128I-T213A-L217N +S024G-G097A-S101Q-S128A-S188D-M222S + S024G-S078N-G097A-S101N +S024G-S078N-G097S-S101G-M222S +G020K-N062E-S078N-N116L-S128Q-T213A-M222S + G020K-N062E-S128Q-M222S +G020K-N062E-S101Q-N116L + G020K-N062E-S101Q-Q109G-N116L-S188D-L217Q +G020K-S024G-N062E-G097A-S101N-N116L-S128A-S188D +G020K-S024G-N062E-S078N-S101Q-Q109G-N116L-S188D-M222S +S078N-G097S-S101N-Q109G-S128A-L217Q-M222S +S024G-S078N-S101N-Q109G-S128A-L217Q-M222S + G097A-S101Q-S128A-S188D +S024G-S078N-G097A-S101N-Q109N-S128A-S188D-L217Q-M222S +S024G-S078N-G097A-S101G-L217E-M222S + S078N-G097S-S101Q-Q109G-T274I +G097A-S101G-M222S + S101N-Q109N + G020K-L217E-S240R-E271L +S128Q-S240R-E271L + G020K-S101D-N116L-S128Q-M222S +G020K-P055L-N062E-N116L-S128I + G020K-N062E-S101G-N116L-S128A-M222S +G020K-S078N-S101D-S128L-S240R-E271L + G020K-T213A +G020K-N062E-S078N-G097S-S101Q-Q109N-N116L-L217Q-M222S +S078N-S101D-S240R-E271L + S024G-S078N-S101N-Q109G-L217Q-M222S +G097A-S101G-Q109G-L217Q-M222S +G020K-S024G-N062E-S078N-G097S-S101N-N116L-S128A-L217Q- + M222SG020K-S078N-S128L-E271L + G020K-N062E-S078N-N116L-L217N +G097A-S101G-Q109G-S188D-L217Q-M222S +S024G-S078N-G097S-S101N-Q109G-S128A +G020K-S024G-N062E-S078N-G097S-S101N-N116L-S188D-M222S +G020K-S024G-N062E-G097S-S101Q-N116L-S128A-M222S +G020K-N062E-S078N-G097A-S101G-Q109G-N116L-S128A-S188D- + L217QG020K-N062E-S101G-N116L-S128A-L217Q-M222S +S024G-G097A-S101Q-S188D-M222S + S024G-S101G + I072V-S101Q-S128A +S078N-G097S-S101N-Q109N + S024G-G097A-S101N-Q109N-S188D-L217Q-M222S +S078N-S101Q-Q109N-S188D-L217Q-M222S + G020K-N043R-N062E-S128L-L217E +N043R-G102A-S128L-S240R + G020K-N116L-N123G-T213A-M222S +G020K-S101D-S128Q-T213A-M222S + S024G-G097A-S101Q-Q109G + G020K-S078N +G020K-N062E-S101Q-N116L-S188D +G020K-N062E-S078N-G097S-S101N-Q109G-N116L-S188D-L217Q- + M222SG020K-S024G-N062E-G097S-S101G-N116L-S128A-L217Q-M222S +G020K-N062E-G097A-S101N-N116L-S128A-L217Q-M222S +S078N-G097S-S101G-Q109N-M222S + S024G-G097A-S101Q-Q109N +S024G-G097S-S101N-Q109N-S128A-S188D-L217Q-M222S +S078N-S101N-Q109N-S188D-L217Q-M222S + S078N-S101D-S128L-L217E-S240R +G020K-N062E-S101D-N116L-S128L + G097S-S101N-S188D-M222S +G020K-S078N-S128L-S240R-E271L + G020K-N062E-N116L-S128I-T213A +G020K-S024G-N062E-S078N-G097S-S101Q-N116L-S188D-M222S +G020K-N062E-S101Q-Q109G-N116L-L217Q-M222S +G020K-S024G-N062E-G097A-S101Q-N116L-L217Q-M222S +G020K-N062E-S078N-G097S-S101Q-N116L-S128A-S188D-M222S- + A232TG020K-S024G-N062E-S078N-G097S-S101Q-Q109N-N116L-S128A- + L217Q-M222SG020K-N062E-G097A-S101Q-N116L-S188D-M222S +S024G-S078N-G097S-S101N-Q109G-S128A-L217Q-M222S +S024G-G097A-S101Q-Q109N-S128A-S188D-L217Q-M222S + G020K-N043R +G020K-N043R-S078N-G102A-S128L + G097R-S101D +G020K-N062E-S078N-I107V-N123G-S128I +G020K-N062E-S078N-N116L-N123G-T213A + G020K-N062E-N116L-S128I-M222S +G020K-N043R-S240R + S078N-S101D-S128L + G020K-S078N-S128Q-E271L +S078N-S101G-S128A-S188D-L217Q-M222S +S078N-G097S-S101G-Q109N-S128A-L217Q-M222S + S240R +G020K-N062E-N116L-S128Q-T213A-M222S +

TABLE 2-4 BMI cleaning performance of Group C1 variants. PI =Performance Index PI > or = 1.50 is +++; PI between 1.49 and 1.20 = ++;PI between 1.19 and 1.1 = +in Detergent Composition 106 as described inTable 1 PI Relative to GG36; BMI assay, Detergent Sequence of GG36Variants (BPN′ numbering) 106 at 25° C.G020R-T022W-S024R-R045T-S101A-N204S-G211Q +++T022W-S024R-S101A-P210I-T213A +++ G020R-S101A-G211Q +++G020R-S024R-S101A-P210I-G211Q-T213A +++ G020R-S101A-P210I-T213A +++T022W-S024R-N043R-S101A-P210I +++ G020R-S024R-N043R-R045T-S101A-G211Q+++ T022R-S101T-S103N-V104L-A232M-Q245R +++G020R-T022A-S024R-R045T-S101A-T213A +++T022R-S101G-S103N-V104I-A232M-Q245R +++G020R-T022W-S024R-S101A-N116L-G211Q-T213A +++G020R-T022W-N043R-S101A-N116L-G211Q +++ G020R-T022W-N043R-R045T-SI0IA+++ T022W-N043R-R045T-S101A-P210I-T213A +++G020R-S024R-R045T-S101A-Q109R-P210I-G211Q-T213A +++S024R-N043K-S101A-N204D-P210I +++ G020R-S024R-R045T-S101A-P210I +++S024R-N043R-R045T-S101A-P210I-T213A +++ G020R-S024R-R045T-S101A-T213A+++ T022R-S101A-S103N-V104I-A232T-Q245R +++G020R-T022W-S024R-R045T-S101A-N204S-P210I-G211Q +++T022W-N043R-S101A-P210I-G211Q +++G020R-S024R-N043K-R045T-S101A-N116L-P210I +++T022A-S101G-S103A-V104I-I107V-L217Q-A232V-Q245R +++S024R-N043R-R045T-S101A-G211Q-T213A +++T022R-S101A-S103G-V104I-A232M-Q245R +++T022R-S101G-S103N-V104L-A232V-Q245R +++S024R-S078G-G118S-Q245R-N248D-H249R +++ S024R-N043R-S101A-P210I-T213A+++ G020R-S024R-N043R-R045T-S101A-T213A +++T022K-S101N-S103A-V104I-A232T-Q245R +++ G020R-N043R-S101A-P210I-G211Q+++ T022R-S101T-S103A-V104I-A232M-Q245R +++S024R-V026A-N043R-S101A-P210I-G211Q +++T022A-S078N-S101G-S103A-V104I-S128A-L217Q-A232V-Q245R +++T022K-S101T-S103G-V104I-A232L-Q245R +++G020R-T022W-S024R-N043R-R045T-S101A-G211Q +++G020R-S024R-S078G-G118D-Q245R +++T022A-S101N-S103A-V104I-S128A-P129S-A232V-Q245R +++T022A-G097A-S101G-S103A-V104I-L217Q-A232V-Q245R +++G020R-T022W-N043R-S101A-G211Q-T213A +++N018K-G020R-S024R-R045T-S101A-P210I-G211Q-T213A +++G020R-T022W-S024R-N043R-R045T-S101A +++T022R-S101N-S103A-V104L-A232T-Q245R +++ G020R-S024R-S101A-T213A +++T022R-S101N-S103G-V104I-A232V-Q245R +++G020R-T022W-S024R-S101A-G211Q-T213A +++T022A-S024G-S101G-S103A-V104I-S128A-L217Q-A232V-Q245R +++S024R-S078G-G118D-P129E-Q245R-H249R +++T022R-S101T-S103A-V104L-A232L-Q245R +++ G020R-S024R-S101A-G211Q-T213A+++ T022A-S024G-S101G-S103A-V104I-L217Q-A232V-Q245R +++G020R-T022W-N043R-V051A-S101A-P210I-G211Q +++ S024R-S078G-G118D-Q245R+++ N043R-S101A-P210I +++ T022Y-S101G-S103N-V104I-A232L-Q245R +++G020R-T022W-N043R-S101A-T213A +++G020R-S024R-S078D-G118S-S188D-Q245R-H249R +++T022Y-S101N-S103N-V104I-A232T-Q245R +++G020R-T022W-S024R-F050L-S101A-G211Q-T213A +++N018S-T022W-S024R-N043R-S101A-G211Q +++T022A-S101G-S103A-V104I-S128A-P129S-L217Q-A232V-Q245R +++T022R-S101N-S103N-V104I-A232V-Q245R +++T022A-S024G-S078N-G097S-S101Q-S103A-V104I-L217Q-A232V- +++ Q245RS101A-P210I-G211Q-T213A +++ G020R-N043R-R045T-S101A-P210I-G211Q-T213A+++ T022R-S101A-S103N-V104I-A232M-Q245S +++G020R-T022W-N043R-R045T-S101A-G211Q +++G020R-T022W-N043R-R045T-S101A-N116L-P210I +++G020R-S024R-S078D-G118S-Q245R +++G020R-T022W-S024R-N043R-S101A-P210I-G211Q-T213A +++G020R-S024R-N043R-R045T-V051A-S101A-P210I-G211Q +++T022A-S101G-S103A-V104I-P129S-L217Q-A232V-Q245R +++G020R-S078G-G118D-Q245R-N248D-H249R +++T022K-S101A-S103N-V104L-A232T-Q245R +++T022A-N076D-S078N-G097S-S101G-S103A-V104I-L217Q-A232V- +++ Q245RS024R-N043R-R045T-S101A-P210I +++T022A-S024G-G061R-S078N-S101G-S103A-V104I-S128A-A232V- +++ Q245RT022A-S078N-S101G-S103A-V104I-L217Q-A232V-Q245R +++T022A-S024G-S078N-S101Q-S103A-V104I-S128A-L217Q-A232V- +++ Q245RT022Q-S101T-S103A-V104L-A232V-Q245R +++G020R-T022W-S024R-R045T-S101A-G211Q +++ P014R +++T022R-S101A-S103N-V104L-A232M-Q245R +++T022K-S101G-S103N-V104I-A232V-Q245R +++ G020R-S024R-S078D-G118D-Q245R+++ T022A-G097S-S101N-S103A-V104I-L217Q-A232V-Q245R +++G020R-S024R-S101A-P210I-T213A +++ T022K-S101N-S103N-V104I-A232V-Q245R+++ T022Y-S101T-S103A-V104I-A232L-Q245R +++T022A-N076D-S078N-S101G-S103A-V104I-S128A-L217Q-A232V- +++ Q245RG020R-S024R-N043R-R045T-S101A-P210I-G211Q-T213A +++G020R-R045T-S101A-P210I-T213A +++ G020R-S078D-G118S-Q245R-H249R +++G020R-T022W-R045T-V051A-S101A +++ T022R-S101T-S103A-V104I-A232V-Q245R+++ N043R-S101A +++ G020R-S024R-N043R-R045T-S101A-G211Q-T213A +++T022R-S101G-S103G-V104I-A232L-Q245R +++ G020R-S101A-P210I +++T022K-S101T-S103N-V104I-A232V-Q245R +++ G020R-T022W-N043R-V051A-S101A+++ G020R-T022W-N043R-S101A +++ G020R-T022W-S024R-N043R-S101A-P210I +++G020R-T022W-S024R-N043R-R045T-S101A-P210I-T213A +++G020R-N043R-R045T-S101A-T213A +++T022A-S024G-S078N-S101N-S103A-V104I-L217Q-A232V-Q245R +++T022Y-S101N-S103N-V104L-A232T-Q245R +++ G020R-S101A-N116L-P210I-G211Q+++ T022R-S101A-S103N-V104L-A232L-Q245R +++ G020R-S024R-N043R-S101A +++T022R-S101G-S103A-V104L-A232T-Q245R +++ G020R-S024R-S101A-G211Q +++T022A-S101Q-S103A-V104I-P129S-A232V-Q245R +++T022A-S024G-G097A-S101G-S103A-V104I-L217Q-A232V-Q245R +++T022A-S024G-S078N-S101G-S103A-V104I-S128A-L217Q-A232V- +++ Q245RT022K-S101A-S103N-V104I-A232T-Q245R +++ G020R-T022W-S024R-R045T-S101A+++ T022Y-S101G-S103A-V104I-A232V-Q245R +++S024R-N043R-R045T-S101A-N116L-P210I-G211Q-T213A +++T022A-S024G-S078N-S101N-S103A-V104I-S128A-L217Q-A232V- +++ Q245RG020R-N043R-S101A-P210I +++T022A-S024G-S078N-G097A-S101G-S103A-V104I-L217Q-A232V- +++ Q245RT022R-S101T-S103N-V104I-A232V-Q245R +++ P055R +++T022A-S101N-S103N-V104I-A232V-Q245R +++G020R-T022W-S024R-N043R-N076D-S101A-P210I-T213A +++G020R-T022W-S101A-P210I-T213A +++ T022Q-S101G-S103A-V104L-A232L-Q245R+++ T022W-S024R-N043R-R045T-S101A +++T022A-S101G-S103A-V104I-L217Q-A232V-Q245R +++T022K-S101N-S103A-V104I-A232L-Q245R +++T022R-S101T-S103N-V104I-A232L-Q245R +++G020R-S024R-N043R-R045T-S101A-P210I-T213A +++T022A-S078N-S101N-S103A-V104I-S128A-A232V-Q245R +++T022A-G097S-S101G-S103A-V104I-L217Q-A232V-Q245R +++T022A-S078N-G097S-S101G-S103A-V104I-L217Q-A232V-Q245R +++G020R-S024R-N062E-S078G-G118S-Q245R +++T022K-S101A-S103N-V104I-A232L-Q245R +++G020R-T022W-S024R-R045T-S101A-P210I-T213A +++G020R-S024R-S078G-G118S-S188D-Q245R +++T022R-S101T-S103A-V104I-A232V-Q245W +++T022R-S101N-S103A-V104I-A232V-Q245R +++T022K-S101T-S103N-V104I-A232I-Q245R +++T022A-S024G-S078N-S101Q-S103A-V104I-L217Q-A232V-Q245R +++T022A-S078N-G097A-S101N-S103A-V104I-A232V-Q245R +++T022Q-S101G-S103N-V104I-A232L-Q245R +++T022A-S078N-G097A-S101Q-S103A-V104I-Q109N-A232V-Q245R +++T022Q-S101G-S103A-V104L-A232V-Q245R +++T022A-S101G-S103A-V104I-S128A-L217Q-A232V-Q245R +++G020R-T022W-N043R-R045T-S101A-P210I +++T022R-S101N-S103N-V104I-A232T-Q245R +++G020R-N043R-R045T-S101A-N116L-G211Q-T213A +++T022Q-S101A-S103N-V104I-A232V-Q245R +++T022W-S024R-N043R-R045T-S101A-N116L-P210I +++T022A-S078N-G097S-S101N-S103A-V104I-L217Q-A232V-Q245R +++G020R-S078D-G118D-Q245R-H249R +++G020R-S024R-N043R-S101A-P210I-G211Q-T213A +++T022R-S101G-S103N-V104I-A232V-Q245W +++T022A-S101T-S103N-V104L-A232L-Q245R +++T022A-S024G-S078N-G097S-S101N-S103A-V104I-S128A-L217Q- +++ A232V-Q245RT022R-S101T-S103N-V104I-A232I-Q245R +++ G020R-N043R-S101A-P210I-T213A+++ G020R-S024R-N043R-S101A-N116L-G211Q-T213A +++S024R-N043R-R045T-S101A-N116L-P210I-G211Q +++T022A-S024G-G097S-S101N-S103A-V104I-L217Q-A232V-Q245R +++ E271R +++T022K-S101T-S103G-V104I-A232V-Q245R +++ N043R-S101A-P210I-G211Q +++G020R-S024R-S078G-G118S-P129E-G159D-Q245R +++T022R-S101T-S103A-V104I-A232T-Q245S +++ E271W +++S024R-N043R-R045T-S101A-N116L-P210I-T213A +++T022A-S024G-S078N-S101N-S103A-V104I-A232V-Q245R +++T022A-E089G-G097A-S101N-S103A-V104I-S128A-A232V-Q245R +++T022R-S101T-S103A-V104I-A232L-Q245R +++T022R-S101G-S103N-V104I-A232T-Q245S +++T022A-S101G-S103A-V104I-A232M-Q245R +++ G020R-T022W-S024R-S101A-T213A+++ G020R-T022W-S024R-R045T-S101A-P210I +++G020R-T022W-S024R-N043R-S101A-T213A +++T022A-S024G-G097S-S101G-S103A-V104I-L217Q-A232V-Q245R +++T022A-S024G-S078N-G097S-S101Q-S103A-V104I-S128A-L217Q- +++ A232V-Q245RG020R-S101A-T213A +++ E271S +++ G020R-S078G-G118S-Q245R-N248D-H249R +++N043K +++ T022A-S101G-S103N-V104I-A232V-Q245R +++T022Q-S101A-S103A-V104I-A232L-Q245R +++G020R-T022W-S024R-R045T-S101A-T213A +++G020R-S024R-S078G-G118S-Q245R-N248D-H249R +++T022A-S024G-S078N-G097A-S101G-S103A-V104I-S128A-L217Q- +++ A232V-Q245RT022A-S024G-S078N-S101G-S103A-V104I-L217Q-A232V-Q245R +++T022A-S024G-S101N-S103A-V104I-A232V-Q245R +++G020R-T022W-S024R-S101A-N116L-P210I +++G020R-T022W-N043R-R045T-S101A-T213A +++T022A-S078N-G097A-S101N-S103A-V104I-L217Q-A232V-Q245R +++T022A-S078N-S101N-S103A-V104I-L217Q-A232V-Q245R +++T022R-S101N-S103G-V104L-A232V-Q245R +++G020R-T022W-S024R-S101A-P210I-T213A +++G020R-S024R-S078D-G118S-S188D-Q245R +++G020R-S024R-S078D-G118S-G159D-Q245R +++T022Y-S101T-S103A-V104I-A232M-Q245R +++G020R-N043R-R045T-V051A-S101A-P210I-G211Q-T213A +++G020R-T022W-S024R-N043R-R045T-S101A-T213A +++T022A-S024G-S078N-G097S-S101G-S103A-V104I-L217Q-A232V- +++ Q245RT022A-S078N-S101Q-S103A-V104I-L217Q-A232V-Q245R +++G020R-S024R-S101A-P210I-G211Q +++ T022Q-S101T-S103G-V104I-A232M-Q245R+++ G020R-N043R-S101A-G211Q-T213A +++T022A-S101Q-S103A-V104I-P129S-L217Q-A232V-Q245R +++T022A-S101T-S103N-V104I-A232V-Q245R +++G020R-S024R-S078G-G118D-G159D-Q245R-N248D-H249R +++G020R-T022W-R045T-S101A-P210I-G211Q +++T022A-S078N-S101N-S103A-V104I-A232V-Q245R +++T022R-S101T-S103N-V104I-S105G-A232V-Q245R +++T022A-S024G-S078N-S101Q-S103A-V104I-A232V-Q245R +++T022A-S101T-S103N-V104I-A232L-Q245R +++T022Q-S101N-S103N-V104L-A232V-Q245R +++T022R-S101N-S103N-V104L-A232M-Q245R +++T022Q-S101G-S103N-V104I-A232T-Q245R +++T022A-S101Q-S103A-V104I-L217Q-A232V-Q245R +++T022Q-S101A-S103A-V104L-A232L-Q245R +++G020R-N043R-R045T-S101A-G211Q-T213A +++T022A-G097A-S101N-S103A-V104I-P129S-L217Q-A232V-Q245R +++G020R-S024R-R045T-S101A-G211Q-T213A +++T022A-S078N-G097A-S101G-S103A-V104I-S128A-A232V-Q245R +++G020R-T022W-N043R-R045T-S101A-P210I-G211Q-T213A +++T022R-S101N-S103N-V104I-A232L-Q245R +++T022Y-S101G-S103A-V104I-A232M-Q245R +++T022R-S101G-S103G-V104I-A232T-Q245R +++T022A-S024G-S078N-G097S-S101N-S103A-V104I-L217Q-A232V- +++ Q245RG020R-S024R-S078G-G118D-N248D-H249R +++T022A-S101A-S103N-V104L-A232V-Q245R +++ T022W-S024R-S101A-N116L-G211Q+++ G020R-S024R-S101A-T208A-P210I-G211Q +++T022W-S024R-N043R-R045T-S101A-P210I-G211Q-T213A +++T022Y-S101G-S103N-V104I-A232M-Q245R +++T022R-S101T-S103A-V104L-A232T-Q245S +++G020R-S024R-S078G-G118S-P129E-Q245R-N248D +++T022A-S024G-S101Q-S103A-V104I-Q109N-A232V-Q245R +++ G020R-T022W-S101A+++ T022W-S024R-N043R-S101A-P210I-G211Q +++G020R-S024R-N043R-S101A-P210I-G211Q +++G020R-S024R-N062E-S078G-G118D-H249R +++ G020R-S024R-G047C-S101A-G211Q+++ G020R-S024R-S078D-G118D-P129E-Q245R-H249R +++T022A-S101A-S103A-V104L-A232V-Q245R +++T022A-S101T-S103N-V104L-A232M-Q245R +++N018S-G020R-T022W-N043R-S101A-G211Q +++T022Y-S101T-S103G-V104I-A232T-Q245R +++T022K-S101A-S103A-V104I-A232M-Q245R +++T022A-S024G-G097A-S101N-S103A-V104I-L217Q-A232V-Q245R +++T022Y-S101N-S103N-V104L-A232V-Q245R +++T022A-S101G-S103A-V104I-A232V-Q245R +++T022A-G097A-S101G-S103A-V104I-P129S-A232V-Q245R +++G020R-S078G-G118S-P129E-Q245R-H249R +++T022A-G097S-S101G-S103A-V104I-S128A-L217Q-A232V-Q245R +++T022Y-S101A-S103G-V104I-A232M-Q245R +++ S024R-S101A-G211Q +++T022A-S024G-G097A-S101Q-S103A-V104I-Q109G-A232V-Q245R +++T022A-S024G-S101G-S103A-V104I-S128A-A232V-Q245R +++R045T-S101A-I107V-Q109R-P210I-G211Q-T213A +++T022A-G097S-S101Q-S103A-V104I-L217Q-A232V-Q245R +++T022K-S101T-S103A-V104I-A232T-Q245R +++Q012R-T022A-S101G-S103A-V104I-N116L-Y209A-T213A-A232V +++T022W-S024R-N043R-R045T-S101A-P210I +++T022A-S101N-S103A-V104I-A232V-Q245R +++T022A-S024G-S078N-G097A-S101N-S103A-V104I-A232V-Q245R +++G020R-S024R-S078G-G118S-G159D +++ T022Y-S101T-S103A-V104I-A232T-Q245R+++ T022Q-S101G-S103N-V104I-A232V-Q245R +++ S024R-S101A-P210I-T213A +++T022A-S101N-S103A-V104I-L217Q-A232V-Q245R +++T022A-S101G-S103N-V104L-A232L-Q245R +++T022A-S024G-G097S-S101N-S103A-V104I-S128A-A232V-Q245R +++T022A-S101G-S103A-V104I-S128A-P129S-A232V-Q245R +++T022A-S101N-S103A-V104L-A232T-Q245R +++ S024R-S101A-P210I +++T022Y-S101T-S103N-V104I-A232V-Q245R +++ G020R-T022W-N043R-S101A-N116L+++ T022Q-S101N-S103N-V104I-A232V-Q245R +++ S024R-S101A +++T022A-S024G-S078N-S101Q-S103A-V104I-Q109N-S128A-A232V- +++ Q245RS024R-N043R-R045T-S101A-N116L-G211Q-T213A +++ S212W +++T022K-S101G-S103A-V104L-A232M-Q245R +++T022A-S024G-S101Q-S103A-V104I-L217Q-A232V-Q245R +++S024R-N043R-R045T-S101A +++ G020R-S101A +++G020R-S024R-S078G-G118D-P129E-Q245R-N248D-H249R +++T022R-S101T-S103N-V104I-A232M-Q245R +++ S056K +++T022A-S024R-N076D-S101G-S103A-V104I-A232V-Q245R-A270V +++T022R-S101T-S103G-V104I-A232M-Q245S +++G020R-T022W-R045T-S101A-P210I-G211Q-T213A +++T022K-S101N-S103N-V104L-A232L-Q245R +++T022W-S024R-N043R-R045T-S101A-V147I-P210I-G211Q-T213A +++T022A-S101Q-S103A-V104I-Q109G-S128A-A232V-Q245R +++T022A-S078N-G097S-S101Q-S103A-V104I-L217Q-A232V-Q245R +++G020R-S024R-S078D-G118S-P129E-Q245R-N248D-H249R +++S078G-G118D-Q245R-H249R +++T022A-S101G-S103A-V104I-A232V-N238Y-Q245R-A272V +++T022A-S024G-S101Q-S103A-V104I-A232V-Q245R +++T022A-S078N-G097S-S101Q-S103A-V104I-A232V-Q245R +++G020R-T022W-S024R-N043R-S101A-P210I-T213A +++T022A-S024G-S101N-S103A-V104I-S128A-L217Q-A232V-Q245R +++ S056R +++T022Y-S101T-S103N-V104I-A232L-Q245R +++T022R-S101T-S103G-V104I-A232V-Q245S +++ T022W-S101A-P210I-G211Q +++T022R-S101A-S103N-V104L-A232T-Q245S +++T022Y-S101A-S103G-V104I-A232T-Q245R +++T022R-S101T-S103A-V104L-A232T-Q245R +++T022Q-S101G-S103A-V104I-A232T-Q245R +++G020R-S024R-S078G-G118D-S188D-Q245R-H249R +++V030I-N043R-S101A-G211Q-T213A +++ T022A-S101T-S103A-V104I-A232V-Q245R+++ T022A-S024G-S101N-S103A-V104I-S128A-A232V-Q245R +++T022A-S101G-S103A-V104I-A232V-N238Y-Q245R +++G020R-S078G-G118D-P129E-G159D-Q245R-H249R +++T022R-S101G-S103A-V104I-A232V-Q245R +++S024R-S078D-G118S-S188D-Q245R-H249R +++T022A-S101G-S103A-V104I-A232V-Q245R-A270V +++T022A-S024G-G097A-S101Q-S103A-V104I-A232V-Q245R +++T022A-S101T-S103G-V104I-A232V-Q245R +++ G020R-T022W-S101A-T213A +++T022A-S101G-S103A-V104I-S128A-A232V-Q245R +++T022A-S101N-S103A-V104I-Q109G-A232V-Q245R +++T022Q-S101N-S103N-V104L-A232L-Q245R +++ N018S-G020R-S101A-P210I-G211Q+++ G020R-S078D-G118S-S188D-Q245R-H249R +++T022A-S101G-S103N-V104I-A232L-Q245R +++T022R-S101T-S103G-V104L-A232V-Q245R +++T022A-N076D-S101G-S103A-V104I-A232V-Q245R-A270V-A272V +++T022Q-S101N-S103G-V104I-A232V-Q245S +++ G020R-N043R-R045T-S101A-G211Q+++ T022A-N076D-S101G-S103A-V104I-L217Q-A232V-Q245R +++T022R-S101A-S103A-V104L-A232V-Q245S +++T022A-S078N-S101Q-S103A-V104I-Q109N-A232V-Q245R +++S024R-S078G-G118S-T180N-Q245R-N248D +++ V004M +++T022A-S101T-S103N-V104I-A232T-Q245R +++T022A-S078N-S101G-S103A-V104I-A230T-A232V-Q245R +++T022Y-S101N-S103N-V104I-A232L-Q245R +++G020R-S024R-S078D-G118S-G159D-Q245R-N248D +++G020R-S024R-S078G-G118S-G159D-S188D-Q245R +++G020R-N043R-R045T-S101A-P210I-T213A +++ S024R-S078D-G118D-Q245R-H249R+++ G020R-S024R-N043R-R045T-S101A-Q109R-T213A +++T022A-S078N-S101N-S103A-V104I-Q109N-A232V-Q245R +++G020R-S024R-N043R-S101A-N204D-G211Q-T213A +++T022A-G097A-S101Q-S103A-V104I-A232V-Q245R +++G020R-S024R-S078G-G118S-N248D +++G020R-S024R-S078G-G118S-G159D-Q245R-N248D +++T022A-S024G-S078N-S101N-S103A-V104I-S128A-A232V-Q245R +++S024R-S078G-G118D-G159D-Q245R-H249R +++T022K-S101T-S103N-V104L-A232V-Q245R +++ S024R-S078G-G118S-Q245R +++T022A-S101A-S103G-V104I-A232L-Q245R +++T022A-G097A-S101Q-S103A-V104I-S128A-A232V-Q245R +++T022A-S024G-S078N-G097S-S101G-S103A-V104I-A232V-Q245R +++G020R-S078D-G118S-Q245R +++T022A-S024R-N076D-S101G-S103A-V104I-A232V-N238Y-Q245R- +++ A270V-A272VS101A-P210I-T213A +++T022A-S101Q-S103A-V104I-S128A-P129S-L217Q-A232V-Q245R +++ A114L +++A016S-T022A-S101G-S103A-V104I-N116A-Y209V-G211Q-T213A- +++ A232VT022A-S101N-S103A-V104I-S128A-P129S-L217Q-A232V-Q245R +++T022R-S101A-S103N-V104I-A232L-Q245S +++T022Q-S101N-S103A-V104I-A232M-Q245W +++T022W-S024R-S101A-N204S-G211Q-T213A +++T022A-S101N-S103A-V104L-A232M-Q245R +++T022A-S024G-S101Q-S103A-V104I-Q109N-S128A-A232V-Q245R +++G020R-S078D-G118S-P129E-Q245R-H249R +++T022Q-S101T-S103A-V104I-A232T-Q245S +++S024R-N043R-R045T-S101A-P210I-G211Q-T213A +++T022R-S101N-S103G-V104I-A232T-Q245R +++ S024R-S101A-P210I-G211Q-T213A+++ T022A-S024G-N076D-G097A-S101G-S103A-V104I-S128A-A232V- +++ Q245RG020R-S024R-S078G-G118S-S188D-N248D-H249R +++T022A-G097S-S101Q-S103A-V104I-Q109G-S128A-A232V-Q245R +++G020R-S024R-R045T-S101A-P210I-T213A +++T022A-S078N-G097S-S101N-S103A-V104I-S128A-A232V-Q245R +++T022A-S024G-S078N-G097A-S101Q-S103A-V104I-A232V-Q245R +++T022R-S101G-S103N-V104I-A232T-Q245W +++T022K-S101G-S103A-V104I-A232V-Q245R +++ T022W-S024R-S101A-G211Q-T213A+++ S024R-N043R-S101A-P210I-G211Q +++T022Q-S101T-S103G-V104I-A232T-Q245R +++T022A-S101G-S103A-V104I-A232V-Q245R-A272V +++T022K-S101G-S103N-V104L-A232M-Q245R +++T022A-S024G-G097A-S101Q-S103A-V104I-Q109N-A232V-Q245R +++S078D-G118S-G159D-Q245R-H249R +++G020R-T022W-S024R-A069T-S101A-P210I-T213A +++T022A-S101N-S103A-V104I-S128A-L217Q-A232V-Q245R +++G020R-T022W-S024R-N043R-R045T-S101A-P210I-G211Q +++G020R-S024R-S078D-G118D-G159D-S188D-Q245R-H249R +++T022A-S078N-S101N-S103A-V104I-S128A-L217Q-A232V-Q245R +++G020R-S078G-G118D-G159D-Q245R-N248D-H249R +++ T022W-R045T-S101A-T213A+++ T022Y-S101N-S103A-V104I-A232T-Q245R ++G020R-R045T-S101A-P210I-G211Q-L267F ++T022Y-S101N-S103N-V104L-A232M-Q245R ++T022A-P052T-S101G-S103A-V104I-L217Q-A232V-Q245R ++T022A-S101G-S103N-V104L-A232T-Q245R ++T022Y-S101A-S103N-V104L-A232V-Q245R ++T022A-S101A-S103N-V104I-A232T-Q245R ++T022Y-S101A-S103N-V104I-A232L-Q245R ++T022Y-S101G-S103A-V104I-A232T-Q245R ++T022A-S024G-N076D-S078N-G097S-S101N-S103A-V104I-L217Q- ++ A232V-Q245RT022A-S101N-S103N-V104L-A232M-Q245R ++T022A-S101Q-S103A-V104I-S128A-P129S-A232V-Q245R ++T022A-S101N-S103A-V104I-S128A-A232V-Q245R ++S024R-S078D-G118S-G159D-Q245R-H249R ++T022K-S101N-S103A-V104L-A232T-Q245R ++T022R-S101T-S103N-V104I-A232T-Q245S ++T022Q-S101N-S103N-V104I-A232M-Q245R ++ S024R-S078G-G118D-Q245R-H249R ++T022R-S101N-S103N-V104L-A232V-Q245R ++T022A-N076D-S101Q-S103A-V104I-Q109G-A232V-Q245R ++G020R-S024R-S078G-G118D-S188D-Q245R-N248D ++T022Y-S101N-S103A-V104I-A232V-Q245R ++T022Q-S101N-S103A-V104L-A232L-Q245R ++T022Q-S101N-S103N-V104I-A232L-Q245R ++T022A-G097S-S101N-S103A-V104I-S128A-L217Q-A232V-Q245R ++T022R-S101N-S103N-V104I-A232T-Q245S ++G020R-S078D-G118D-G159D-Q245R-H249R ++T022R-S101N-S103A-V104L-A232M-Q245R ++T022A-N076D-G097S-S101Q-S103A-V104I-Q109N-S128A-A232V- ++ Q245RT022R-S101G-S103N-V104L-A232T-Q245S ++G020R-T022W-N043R-R045T-S101A-G211Q-T213A ++T022Q-S101T-S103N-V104I-A232T-Q245R ++G020R-S024R-N043R-S101A-N204D-T213A ++T022A-S024G-S101Q-S103A-V104I-Q109G-A232V-Q245R ++G020R-S024R-S078D-G118D-S188D-Q245R-N248D-H249R ++S024R-N043R-S101A-P210I ++T022A-N076D-S101G-S103A-V104I-A232V-Q245R-A270V ++G020R-S024R-S078D-G118S-H249R ++ S024R-S078G-G118S ++T022R-S101G-S103N-V104L-A232V-Q245S ++ S024R-N043K-R045T-S101A ++T022A-N076D-S078N-S101N-S103A-V104I-A232V-Q245R-T274A ++T022A-S024G-N076D-S078N-S101G-S103A-V104I-L217Q-A232V- ++ Q245RT022Q-S101T-S103G-V104I-A232M-Q245W ++S024R-S078G-G118S-P129E-Q245R-H249R ++T022A-S024R-N076D-S101G-S103A-V104I-A232V-Q245R ++ V026W ++T022R-S101G-S103N-V104I-A232L-Q245W ++ G020R-S024R-S078D-G118S-N248D ++G020R-R045T-S101A-N204S-P210I ++G020R-S024R-S078G-G118D-G159D-N248D-H249R ++T022Y-S101A-S103N-V104I-A232V-Q245R ++T022A-S024G-S078N-G097A-S101N-S103A-V104I-L217Q-A232V- ++ Q245RS024R-S101A-P210I-G211Q ++ T022R-S101N-S103N-V104I-A232V-Q245S ++T022A-S024G-G097A-S101N-S103A-V104I-Q109G-A232V-Q245R ++T022A-G097A-S101N-S103A-V104I-A232V-Q245R ++T022A-S024G-N076D-S101Q-S103A-V104I-Q109N-A232V-Q245R ++G020R-S078G-G118S-H249R ++ S078G-G118S-Q245R-H249R ++T022A-G097A-S101N-S103A-V104I-L217Q-A232V-Q245R ++T022Y-S101T-S103G-V104I-A232M-Q245R ++T022K-S101G-S103N-V104I-A232L-Q245R ++T022A-S024G-G097S-S101N-S103A-V104I-A232V-Q245R ++S024R-S078G-G118D-G159D-S188D-Q245R-H249R ++T022Q-S101N-S103A-V104I-A232V-Q245R ++G020R-S024R-S078G-G118D-G159D-Q245R-N248D ++T022A-S024G-S101G-S103A-V104I-A232V-Q245R ++T022Q-S101G-S103A-V104L-A232T-Q245R ++T022Q-S101T-S103A-V104I-A232T-Q245R ++T022A-N076D-S101N-S103A-V104I-S128A-A232V-Q245R ++T022A-S024G-N076D-S078N-G097A-S101G-S103A-V104I-A232V- ++ Q245RT022A-S101G-S103A-V104L-A232V-Q245R ++T022A-S024G-S078N-G097S-S101Q-S103A-V104I-S128A-A232V- ++ Q245RT022A-S024G-N076D-G097S-S101G-S103A-V104I-A232V-Q245R ++S024R-N043R-S101A-G211Q-T213A ++T022A-G097S-S101Q-S103A-V104I-A232V-Q245R ++S024R-S078G-G118S-Q245R-N248D ++G020R-T022W-S024R-N043R-S101A-N204S-P210I-G211Q ++T022A-S024G-S078N-G097S-S101N-S103A-V104I-Q109G-S128A- ++ A232V-Q245RT022A-S101G-S103A-V104I-P129S-A232V-Q245R ++T022R-S101T-S103G-V104I-A232T-Q245S ++T022A-S024G-I072V-G097S-S101Q-S103A-V104I-S128A-L217Q- ++ A232V-Q245RT022K-S101A-S103A-V104I-A232V-Q245W ++G020R-T022W-S024R-R045T-S101A-N116L-P210I ++T022R-S101T-S103G-V104I-A232T-Q245W ++T022A-S024G-N076D-S078N-G097S-S101N-S103A-V104I-S128A- ++ A232V-Q245RT022A-G097A-S101N-S103A-V104I-S128A-A232V-Q245R ++T022Y-S101T-S103N-V104L-A232V-Q245R ++G020R-S024R-N062E-S078G-G118S-N248D ++T022Q-S101G-S103A-V104I-A232V-Q245R ++T022A-N076D-S078N-S101N-S103A-V104I-S128A-A232V-Q245R ++T022A-S024G-G097A-S101N-S103A-V104I-S128A-A232V-Q245R ++T022A-S024G-N076D-S078N-S101Q-S103A-V104I-A232V-Q245R ++T022A-N076D-S078N-S101G-S103A-V104I-L217Q-A232V-Q245R ++T022A-S024G-N076D-S101N-S103A-V104I-A232V-Q245R ++T022R-S101N-S103A-V104I-A232L-Q245S ++T022A-S078N-S101N-S103A-V104I-Q109N-L217Q-A232V-Q245R ++T022R-S101A-S103A-V104I-A232V-Q245W ++T022A-S101Q-S103A-V104I-A232V-Q245R ++T022A-S078N-G097S-S101N-S103A-V104I-A232V-Q245R ++T022A-G097A-S101Q-S103A-V104I-P129S-A232V-Q245R ++T022A-S101T-S103A-V104I-A232L-Q245R ++T022A-A085T-G097S-S101Q-S103A-V104I-L217Q-A232V-Q245R ++T022A-S101T-S103N-V104L-S105G-A232M-Q245R ++T022A-S101A-S103A-V104I-A232M-Q245S ++T022Q-S101A-S103A-V104I-A232M-Q245R ++T022Q-S101G-S103A-V104L-A232M-Q245R ++T022A-S024G-G097A-S101Q-S103A-V104I-S128A-A232V-Q245R ++T022A-S024G-G097S-S101Q-S103A-V104I-L217Q-A232V-Q245R ++T022A-N076D-S078N-G097S-S101G-S103A-V104I-S128A-A232V- ++ Q245RT022A-G097A-S101G-S103A-V104I-H120Q-S128A-A232V-Q245R ++T022A-G097A-S101N-S103A-V104I-S128A-P129S-L217Q-A232V- ++ Q245RG020R-S024R-N062E-S078G-G118D-Q245R ++T022K-S101G-S103G-V104I-A232V-Q245R ++ S078G-G118S-S188D-Q245R-H249R ++T022W-N043R-S101A-G211Q-T213A ++T022A-S024G-N076D-G097S-S101N-S103A-V104I-Q109N-A232V- ++ Q245RT022Y-S101A-S103N-V104L-A232M-Q245R ++G020R-T022W-S101A-N116L-G211Q-T213A ++T022W-S024R-N043R-R045T-S101A-T213A ++ N043R-S101A-G211Q-T213A ++T022Y-S101N-S103N-V104L-A232L-Q245R ++ T255G ++T022A-G097S-S101N-S103A-V104I-S128A-A232V-Q245R ++T022W-S024R-N043R-S101A-P210I-T213A ++ N269K ++T022R-S101T-S103A-V104I-A232L-Q245S ++T022A-S024G-I072V-S078N-G097S-S101N-S103A-V104I-S128A- ++ A232V-Q245RT022K-S101T-S103G-V104I-A232M-Q245R ++H017R-N043R-R045T-S101A-P210I-T213A ++T022A-S024G-G097S-S101G-S103A-V104I-S128A-A232V-Q245R ++T022A-S078N-G097A-S101Q-S103A-V104I-A232V-Q245R ++T022A-N076D-G097A-S101N-S103A-V104I-A232V-Q245R ++T022A-S024G-V026A-S101G-S103A-V104I-S128A-A232V-Q245R ++T022A-S024G-G097A-S101G-S103A-V104I-S128A-L217Q-A232V- ++ Q245RT022Q-S101T-S103G-V104I-A232L-Q245R ++T022W-S024R-S101A-P210I-G211Q-T213A ++T022Q-S101N-S103A-V104I-A232M-Q245R ++ G020R-R045I-S101A ++T022Y-S101N-S103N-V104I-A232V-Q245R ++T022R-S101G-S103A-V104I-A232L-Q245R ++S024R-S078D-G118D-P129E-Q245R-H249R ++T022R-S101A-S103A-V104I-A232M-Q245S ++ G020R-S101A-P210I-G211Q ++T022W-N043R-R045T-S101A-P210I-G211Q ++T022R-S101G-S103A-V104I-A232M-Q245S ++T022A-S024G-N076D-S101G-S103A-V104I-L217Q-A232V-Q245R ++T022R-S101T-S103N-V104I-A232V-Q245S ++T022Q-S101G-S103N-V104L-A232M-Q245R ++T022K-T038I-S101G-S103A-V104I-A232T-Q245S ++N043R-R045T-S101A-P210I-G211Q ++ T022K-S101T-S103A-V104L-A232M-Q245R ++T022Y-S101T-S103N-V104I-A232T-Q245R ++G020R-S024R-N043R-S101A-P210I-T213A ++ S099G-S101G ++T022A-S101N-S103N-V104L-A232T-Q245R ++T022A-S024G-N076D-S078N-G097A-S101N-S103A-V104I-A232V- ++ Q245RT022R-S101N-S103A-V104I-A232V-Q245W ++ T022W-N043R-R045T-S101A-P210I ++T022K-S101A-S103G-V104I-A232T-Q245R ++T022Q-S101N-S103A-V104I-A232T-Q245S ++T022A-S101Q-S103A-V104I-Q109G-A232V-Q245R ++T022A-G097A-S101Q-S103A-V104I-S128A-P129S-L217Q-A232V- ++ Q245RT022A-S078N-G097A-S101Q-S103A-V104I-L217Q-A232V-Q245R ++S024R-S078G-G118S-G159D-Q245R ++ T022K-S101N-S103N-V104I-A232I-Q245R ++A098F-S099A-V104I ++ G020R-R045T-S101A-G211Q-T213A ++T022A-N076D-S078N-G097A-S101G-S103A-V104I-A232V-Q245R ++S024R-S078D-G118S-G159D-Q245R ++T022A-S024G-N076D-S101N-S103A-V104I-Q109N-A232V-Q245R ++T022K-S101T-S103N-V104I-A232M-Q245R ++T022A-S024G-S078N-G097S-S101N-S103A-V104I-S128A-A232V- ++ Q245RT022Q-S101T-S103N-V104I-A232L-Q245S ++ A114T ++T022Q-S101N-S103N-V104L-A232T-Q245R ++T022A-S078N-S101G-S103A-V104I-Q109N-A232V-Q245R ++T022K-S101T-S103A-V104I-A232M-Q245R ++T022A-S024G-G097S-S101Q-S103A-V104I-Q109G-S128A-L217Q- ++ A232V-Q245R ++T022A-S101G-S103A-V104I-N116L-Y209V-G211Q-A232V ++T022A-S101T-S103N-V104I-A232M-Q245W ++G020R-T022W-S024R-S101A-N116L-G211Q ++T022A-S078N-S101Q-S103A-V104I-S128A-L217Q-A232V-Q245R ++G020R-R045T-S101A-G211Q ++T022A-N076D-S078N-G097A-S101G-S103A-V104I-S128A-A232V- ++ Q245RS024R-S101A-T213A ++ T022A-S078N-S101G-S103A-V104I-A232V-Q245R ++G020R-S024R-S078D-G118S-G159D-S188D-Q245R-N248D-H249R ++T022Y-S101N-S103N-V104I-A232V-Q245W ++T022Y-S101A-S103G-V104I-A232V-Q245R ++T022A-S024R-N076D-S101G-S103A-V104I-A232V-N238Y-Q245R ++G020R-S078G-G118S-GI59D-H249R ++T022A-S024R-S101G-S103A-V104I-P129E-A232V-Q245R-A272V ++T022R-S101N-S103A-V104I-A232V-Q245S ++ G020R-S101A-P210I-G211Q-T213A ++G020R-S024R-S078D-G118S-G159D-S188D-Q245R ++ S087R ++G020R-S024R-R045T-A048T-S101A-G211Q ++T022A-S024G-G097S-S101Q-S103A-V104I-A232V-Q245R ++G020R-S024R-S078G-G118S-P129E-S188D-Q245R-N248D-H249R ++T022A-G097S-S101G-S103A-VI04I-Q109N-A232V-Q245R ++L021S-T022A-S024G-N076D-S078N-S101G-S103A-V104I-S128A- ++ A232V-Q245RS242K ++ T022K-S101A-S103A-V104I-A232V-Q245R ++T022K-S101N-S103A-V104L-A232L-Q245R ++T022A-N076D-S101G-S103A-V104I-A232V-N238Y-Q245R-A270V- ++ A272VT022R-S101T-S103A-V104I-A232V-Q245S ++T022Y-S101N-S103G-V104I-A232L-Q245R ++T022Q-S101N-S103G-V104I-A232V-Q245R ++T022A-S101G-S103A-V104I-A232V-Q245W ++T022R-S101A-S103A-V104I-A232L-Q245S ++G020R-T022W-R045T-F050L-S101A-P210I-G211Q-T213A ++T022A-S101Q-S103A-V104I-S128A-L217Q-A232V-Q245R ++G020R-S101A-G211Q-T213A ++T022A-N076D-S101G-S103A-V104I-A232V-Q245R-A272V ++T022A-S101G-S103A-V104I-A232V-N238Y-Q245R-A270V ++T022A-S101N-S103G-V104I-A232V-Q245R ++ G020R-S101A-T208P-P210I-G211Q ++D181S ++ S101G-S103A-V104I-N116L-Y209V-A232V ++T022Q-S101A-S103N-V104I-A232T-Q245R ++T022A-S101G-S103N-V104L-G211S-A232T-Q245R ++T022A-G097A-S101Q-S103A-V104I-L217Q-A232V-Q245R ++T022A-S101T-S103N-V104I-A232M-Q245S ++T022A-S101G-S103A-V104I-Q109N-A232V-Q245R ++T022Q-S101A-S103G-V104I-A232M-Q245R ++G020R-S078G-G118S-G159D-N248D-H249R ++T022A-N076D-S101N-S103A-V104I-A232V-Q245R ++T022A-S024G-G097S-S101N-S103A-V104I-Q109G-S128A-A232V- ++ Q245RT022Q-S101A-S103A-V104I-A232T-Q245R ++T022A-S101G-S103A-V104I-A232L-Q245R ++T022A-S101G-S103A-V104I-P129E-A232V-N238Y-V244I-Q245R- ++ A270V-A272VG020R-S024R-S078D-G118D-G159D-Q245R-N248D-H249R ++T022R-S101A-S103N-V104I-A232V-Q245W ++T022Q-S101A-S103N-V104I-A232M-Q245R ++T022Q-S101A-S103N-V104L-A232L-Q245R ++T022A-S024G-S101Q-S103A-V104I-S128A-L217Q-A232V-Q245R ++T022Q-S101G-S103G-V104I-A232M-Q245R ++G020R-S078G-G118S-G159D-Q245R-H249R ++T022R-S101A-S103N-V104L-A232V-Q245S ++T022A-S024V-G097S-S101N-S103A-V104I-H120N-S128A-A232V- ++ Q245RT022A-A073V-N076D-S078N-S101N-S103A-V104I-A232V-Q245R ++T022Q-S101G-S103N-V104L-A232V-Q245R-T2741 ++T022A-S101N-S103N-V104I-A232T-Q245R ++T022W-S024R-R045P-S101A-P210I-G211Q-T213A ++G020R-S024R-N062E-S078G-G118D-Q245R-N248D ++T022Q-S101A-S103N-V104L-A232M-Q245R ++T022Q-S101T-S103G-V104L-A232T-Q245R ++ T022R-S101G-S103N-A232L-Q245R ++T022A-G097S-S101Q-S103A-V104I-Q109N-L217Q-A232V-Q245R ++T022A-S024G-G097A-S101Q-S103A-V104I-S128A-L217Q-A232V- ++ Q245RS024R-S101A-G211Q-T213A ++ T022R-S101N-S103N-V104L-A232T-Q245S ++G020R-R045T-S101A-P210I ++ T022R-S101A-S103A-V104I-A232M-Q245W ++T022A-S101N-S103N-V104I-A232L-Q245R ++T022A-S101G-S103G-V104I-A232M-Q245R ++G020R-S024R-S078D-G118S-S188D-Q245R-N248D ++ S099T-S103G ++T022W-N043R-S101A-P210I ++ T022W-A029V-N043R-S101A-P210I ++ F050R ++T022A-S024G-N076D-S078N-G097S-S101Q-S103A-V104I-Q109N- ++ A232V-Q245RT022A-S024G-N076D-S101G-S103A-V104I-A232V-Q245R ++T022R-S101G-S103N-V104L-A232L-Q245R ++T022A-S101N-S103A-V104I-Q109N-S128A-A232V-Q245R ++ S024R-R045T-S101A ++T022A-N076D-G097S-S101N-S103A-V104I-L217Q-A232V-Q245R ++T022Q-S101G-S103A-V104I-A232M-Q245S ++T022W-S024R-N043R-R045T-S101A-G211Q-T213A ++ G020R-S078G-G118S ++T022W-N043R-G046C-S101A-P210I-G211Q ++ S101A-G211Q-T213A ++T022A-N076D-S078N-S101G-S103A-V104I-A232V-Q245R ++T022A-S024G-N076D-G097S-S101Q-S103A-V104I-A232V-Q245R ++T022A-S101N-S103A-V104I-L126I-P129S-A232V-Q245R ++T022R-S101T-S103N-V104I-A232L-Q245W ++T022A-S101G-S103A-V104I-A232V-N238Y-Q245R-A270V-A272V ++S078G-G118D-Q245R ++T022A-S024R-S101G-S103A-V104I-P129E-A232V-N238Y-Q245R- ++ A270VT022A-N076D-G097S-S101N-S103A-V104I-Q109G-S128A-A232V- ++ Q245RT022W-S024R-R045T-S101A-N116L-P210I-T213A ++T022R-S101T-S103N-V104I-A232L-Q245S ++T022W-S024R-N043R-R045T-S101A-S106L-P210I-G211Q ++T022A-E089G-G097A-S101Q-S103A-V104I-P129S-L217Q-A232V- ++ Q245R V104I ++T022A-S101A-S103G-V104I-A232L-Q245S ++ N043R-S101A-P210I-T213A ++N043R-R045T-S101A-T213A ++ S078G-G118D-H120N-H249R ++T022Q-S101T-S103N-V104I-A232L-Q245R-R275H ++T022Y-S101A-S103A-V104I-A232T-Q245S ++T022K-S101N-S103N-V104I-A232M-Q245R ++T022R-S101T-S103N-V104L-A232V-Q245S ++T022A-N076D-G097S-S101N-S103A-V104I-A232V-Q245R ++ M119L ++A098F-S099A-S101G-V104I ++ S078D-G118S-Q245R ++T022Y-S101N-S103A-V104I-A232L-Q2455 ++T022A-G097A-S101N-S103A-V104I-S128A-L217Q-A232V-Q245R ++G020R-S078G-G118D-N248D-H249R ++ T022R-S101A-S103N-V104I-A232T-Q245S ++T022R-S101G-S103N-V104I-A232L-Q245S ++S024R-S078D-G118D-S188D-Q245R-H249R ++ S132H ++ A098Q-S099G-S101G-V104L++ N043R-S101A-P210I-G211Q-T213A ++ T022Q-S101G-S103A-V104I-A232M-Q245W++ T022A-S101Q-S103A-V104I-L126I-P129S-A232V-Q245R ++T022K-S101I-S103G-V104I-A232M-Q245S ++T022W-N043R-R045T-S101A-G211Q-T213A ++T022Q-S101N-S103G-V104I-A232V-Q245W ++ D181T ++G020R-S024R-S078D-G118S-P129E-G159D-Q245R-H249R ++G020R-S024R-S078G-G118D-G159D-S188D-Q245R ++T022A-S101T-S103N-V104L-A232L-Q245S ++T022A-S101G-S103N-V104L-A232V-Q245S ++T022A-S024G-G097A-S101N-S103A-V104I-S128A-L217Q-A232V- ++ Q245RT022A-S024G-N076D-S101N-S103A-V104I-S128A-A232V-Q245R ++T022Y-S101T-S103A-V104L-A232M-Q245R ++T022Q-S101T-S103N-V104L-A232T-Q245R ++ F050S ++T022Y-S101G-S103N-V104I-A232V-Q245R ++ G020R-R045T-S101A-T213A ++T022K-S101N-S103G-V104I-A232V-Q245W ++S024R-S078G-G118D-P129E-G159D-Q245R-H249R ++T022Q-S101T-S103N-V104I-A232M-Q245W ++ S049W ++T022A-S101A-S103N-V104L-A232L-Q245R ++T022A-N076D-S101G-S103A-V104I-I107V-P129G-A232V-Q245R- ++ A270VS099A-V104I ++ T022Q-S101A-S103G-V104I-A232V-Q245W ++T022A-N076D-S101G-S103A-V104I-A232V-N238Y-Q245R ++T022A-S101G-S103A-V104I-L124V-L217Q-A232V-Q245R ++G020R-S024R-S078D-G118D-G159D-H249R +G020R-S024R-S078G-G118S-G159D-S188D-Q245R-N248D-H249R +T022A-S101T-S103G-V104I-A232T-Q245S +T022A-S101I-S103G-V104I-A232T-Q245R +T022Q-S101G-S103N-V104I-A232V-Q245W + T022W-N043R-S101A-T213A +T022A-N076D-S078N-G097A-S101N-S103A-V104I-S128A-A232V- + Q245RT022A-S024G-S078N-S101Q-S103A-V104I-S128A-A232V-Q245R +T022K-S101N-S103N-V104L-A232T-Q245R +T022A-S024G-S101Q-S103A-V104I-S128A-A232V-Q245R +G020R-S024R-N062E-S078G-G118D + G020R-S078G-G118D +T022Y-S101T-S103G-V104L-A232T-Q245R +R010H-T022A-S101G-S103A-V104I-A232V-Q245R-A272V +T022A-S024R-S101G-S103A-V104I-P129E-A232V-Q245R-A270V- + A272VT022A-N076D-S078N-G097S-S101Q-S103A-V104I-Q109G-A232V- + Q245RG020R-S024R-S078D-G118S-P129E-Q245R +T022A-N076D-S101G-S103A-V104I-A232V-N238Y-Q245R-A272V +T022K-S101N-S103N-V104I-A232V-Q245W +T022Q-S101T-S103N-V104I-A232M-Q245S +T022A-S078N-S101N-S103A-V104I-Q109G-L217Q-A232V-G245R + W113Y +T022A-S101A-S103N-V104I-A232M-Q245S +T022A-S101Q-S103A-V104I-S128A-A232V-Q245R +G020R-S024R-S078G-G118D-G159D-S188D-H249R +T022A-S101N-S103G-V104I-A232T-Q245R +T022K-S101G-S103G-V104I-A232L-Q245R +T022A-N076D-G097A-S101Q-S103A-V104I-S128A-A232V-Q245R +T022Q-S101T-S103N-V104I-A232V-Q245S +T022A-S024F-S101G-S103A-V104I-N116L-Y209V-T213A-A232V +T022A-S101N-S103G-V104L-A232T-Q245R +T022A-S024R-S101G-S103A-V104I-P129E-A232V-N238Y-Q245R +T022A-N076D-G097S-S101Q-S103A-V104I-A232V-Q245R +T022A-N076D-S078N-G097A-S101N-S103A-V104I-A232V-Q245R +T022A-S101N-S103A-V104I-Q109G-S128A-A232V-Q245R + S259W +A016S-T022A-S024F-S101G-S103A-V104I-N116A-Y209V-G211Q- + A232VT022W-S101A-P210I-G211Q-T213A +T022A-S024G-N076D-G097S-S101N-S103A-V104I-A232V-Q245R + V028T-A215V +S024R-S078D-G118S-S188D-Q245R + T022Q-S101T-S103A-V104I-A232V-Q245W +T022A-S101N-S103A-V104I-Q109G-L217Q-A232V-Q245R +T022A-S024G-T071A-G097S-S101Q-S103A-V104I-S128A-A232V- + Q245R H120P +G020R-S024R-N062E-S078D-G118D-Q245R +T022A-N076D-S101Q-S103A-V104I-A232V-Q245R +T022Q-S101A-S103N-V104I-A232L-Q245W +T022Q-S101N-S103A-V104L-A232T-Q245R +T022W-S024R-N043R-R045T-S101A-N116L-P210I-G211Q +T022A-G097S-S101Q-S103A-V104I-S128A-L217Q-A232V-Q245R +G020R-S078G-G118S-G159D-S188D-H249R + A013S + A232N +T022A-N076D-S101G-S103A-V104I-A232V-Q245R +T022A-S024G-N076D-S078N-G097S-S101N-S103A-V104I-A232V- + Q245R I035T +V051M + N123A + G020R-S078G-G118S-G159D-Q245R-N248D +T022Y-S101A-S103N-V104I-A232L-Q245W +T022A-S024R-S101G-S103A-V104I-P129E-A232V-Q245R-A270V +G020R-S078D-G118S + T022R-S101N-S103N-V104L-A232M-Q245W +T022A-S024G-N076D-G097S-S101G-S103A-V104I-S128A-L217Q- + A232V-Q245RT022A-S101G-S103N-V104L-A232V-Q245R +T022A-N076D-G097A-S101Q-S103A-V104I-A232V-Q245R +T022A-S024R-N076D-S101G-S103A-V104I-I107V-P129E-A232V- + Q245RS024R-R045T-S101A-P210I-G211Q +T022A-S078N-S101N-S103A-V104I-Q109N-S128A-A232V-Q245R +T022A-S024G-S078N-G097A-S101N-S103A-V104I-Q109N-A232V- + Q245RG020R-S024R-S078G-G118D-P129E-G159D-H249R +G020R-S078G-G118D-P129E-S188D-Q245R-H249R + P014W +G020R-S024R-S078G-G118S-G159D-S188D-Q245R-N248D +T022R-S101G-S103N-V104I-A232M-Q245W +T022R-A098V-S101G-S103N-V104L-A232T-Q245R +T022A-S101T-S103A-V104I-A232V-Q245W + G020R-T022W-R045T-S101A-T213A +N043R-R045T-S101A + T022K-S101N-S103A-V104I-A232V-Q245R +T022K-S101I-S103N-V104I-A232L-Q245S + S056V +G020R-T022W-R045I-S101A-G211Q + G020R-S024R-S078D-G118D-Q245R-N248D +G020R-S024R-S078D-G118S-G159D-V244L-Q245R-N248D +T022A-G097A-S101Q-S103A-V104I-Q109G-S128A-A232V-Q245R +T022A-N076D-G097S-S101G-S103A-V104I-A232V-Q245R +G020R-T022W-R045T-S101A-N116L-P210I +T022Q-S101N-S103N-V104I-A232M-Q245S +T022Y-S101G-S103N-V104I-A232V-Q245W + A114V + S141C +T022A-N076D-S078N-S101N-S103A-V104I-A232V-Q245R + S056H +S078D-G118D-Q245R + S024R-N043R-R045T-S101A-T213A +T022Y-S101T-S103G-V104I-A232V-Q245S +T022A-S024G-G097S-S101Q-S103A-V104I-S128A-L217Q-A232V- + Q245R N269W +S078G-G118S-G159D-Q245R-N248D-H249R + T022W-S024R-R045T-S101A +T022A-S024R-S101G-S103A-V104I-P129E-A232V-N238Y-Q245R- + A270V-A272VT022A-S024R-S101G-S103A-V104I-P129E-A232V-Q245R +T022A-S024R-N076D-S101G-S103A-V104I-I107V-P129E-A232V- +N238Y-Q245R-A270V-A272V A122F +S024R-S078D-G118S-P129E-G159D-Q245R-H249R + S099G +T022K-S101T-S103G-V104I-A232V-Q245S +S009G-T022A-S078N-S101Q-S103A-V104I-Q109G-S128A-L217Q- + A232V-Q245RA098Q + T022A-S101N-S103A-V104L-A232L-Q245R +T022A-S101A-S103N-V104I-A232T-Q245W +T022A-S024G-N076D-G097A-S101N-S103A-V104I-A232V-Q245R +S024R-S078G-G118D-G159D-H249R +T022A-S024G-N076D-G097S-S101N-S103A-V104I-S128A-A232V- + Q245RT022A-S024G-N076D-S078N-S101Q-S103A-V104I-S128A-A232V- + Q245RT022A-S101Q-S103A-V104I-L124V-S128A-P129S-A232V-Q245R +T022R-S101N-S103A-V104I-A232M-W241R-Q245W + R045T-S101A-P210I-G211Q +G020R-S024R-S078G-G118S-P129E-Q245V-N248E +T022Q-S101T-S103N-V104I-A232L-Q245W +T022A-S024G-S078N-S101N-S103A-V104I-Q109G-L217Q-A232V- + Q245RT022A-S024G-G097A-S101N-S103A-V104I-L217Q-A223S-A232V- + Q245RT022K-S101T-S103A-V104I-A232T-Q245W + N043R-S101A-G211Q +T022A-S101G-S103A-V104I-Q109N-S128A-A232V-Q245R +T022A-G097A-S101G-S103A-V104I-S128A-L217Q-A232V-Q245R +G020R-S078G-G118S-P129E-H249R + S024R-S078G-G118S-G159D-S188D-Q245R +N043R-R045T-S101A-N204S-P210I-T213A +T022R-S101N-S103A-V104L-A232V-Q245S +T022R-S101N-S103G-V104I-A232V-Q245S +T022K-S101T-S103N-V104I-A232M-Q245S +T022A-N076D-S101N-S103A-V104I-L217Q-A232V-Q245R +T022A-S024G-N076D-S078N-S101N-S103A-V104I-Q109G-S128A- + A232V-Q245RG020R-T022W-R045T-S101A-P210I-T213A +T022A-N076D-G097A-S101G-S103A-V104I-L217Q-A232V-Q245R +T022W-S101A-P210I +T022A-G097A-S101N-S103A-V104I-L126I-P129S-A232V-Q245R +T022A-S078D-S101N-S103A-V104I-S128A-A232V-Q245R + Q236L +T022Y-S101T-S103N-V104I-A232V-Q245S + A200D +G020R-S078G-G118S-S188D-H249R + T022A-S101G-S103N-V104I-A232T-Q245W +T022A-S024G-N076D-G097A-S101N-S103A-V104I-S128A-A232V- + Q245R G157I +G020R-S024R-S078G-G118S-P129E-G159D-S188D-Q245R +T022Y-S101A-S103G-V104L-A232T-Q245R + S024R-S078G-G118D-H249R +T022Q-S101A-S103N-V104L-A232T-Q245S +T022Y-S101A-S103N-V104I-A232T-Q245W +T022A-S024G-S101N-S103A-V104I-Q109N-L217Q-A232V-Q245R +T022A-S101G-S103N-V104I-A232M-Q245S +T022A-S024G-N076D-S078N-S101G-S103A-V104I-S128A-A232V- + Q245RT022A-S101G-S103N-V104I-A232M-Q245W + I008N +T022A-S101N-S103G-V104I-A232M-Q245S + V026A +T022R-S101N-S103A-V104L-A232T-Q245W +T022A-S101A-S103N-V104I-A232L-Q245S +T022A-S101N-S103A-V104I-A232T-Q245R +N043R-R045T-S101A-P210I-G211Q-T213A +T022A-S024G-N076D-G097A-S101Q-S103A-V104I-S128A-A232V- + Q245RA098F-S099T-S101A + A098F-S099T-S101G-V104L + P014N +A098Q-S103G-V104I + S099T-S103A-V104I + S087W +S024R-N062E-S078G-G118S-H249R + S024R-N043R-R045T-S101A-G211Q +N043R-R045T-S101A-G211Q + G020R-S078G-G118S-N248D +T022Q-S101N-S103N-V104I-A232T-Q245W + S078D-G118S-P129E-Q245R-H249R +T022Q-S101T-S103G-V104I-A232T-Q245S +T022A-S024G-S101N-S103A-V104I-Q109N-S128A-A232V-Q245R +S024F-S101G-S103A-V104I-N116L-Y209A-T213A-A232V +S101A-N116L-P210I-T213A + T022R-S101N-S103N-V104L-A232L-Q245S + B015T +S099T + T022A-S101G-S103A-V104I-L124V-A232V-Q245R +G020R-S024R-S078D-G118D-G159D-Q245R + A114I + S212A +T022A-S101T-S103N-V104I-A232L-Q245S +T022A-S024G-S078N-G097S-S101N-S103A-V104I-S128A-T143I- +L217Q-A232V-Q245R S024R-S078G-G118S-G159D-S188D-Q245R-H249R +T022K-S101A-S103N-V104L-A232L-Q245S + T022W-S101A-T213A +T022A-N076D-S078N-G097S-S101Q-S103A-V104I-A232V-Q245R + S132V +S024R-S078G-G118D-S188D-Q245R-N248D-H249R + A098Q-S101A-S103A-V104L +A098Q-S099T-S103A + T022R-S101T-S103N-V104L-A232T-Q245W +T022A-N076D-S101Q-S103A-V104I-S128A-A232V-Q245R +V051A-S101A-P210I-T213A + A098Q-S099T-S101A-V104L +T022R-S101N-S103N-V104I-A232M-Q245W + S099A-S101A-S103A +G020R-S078G-G118D-S188D-Q245R +G020R-S024R-S078D-G118S-P129E-G159D-S188D-Q245R-H249R +T022A-N076D-S078N-G097A-S101Q-S103A-V104I-L217Q-A232V- + Q245RS024R-S078G-G118D + T022Q-S101N-S103N-V104L-A232M-Q245R +T022A-S101T-S103G-V104I-A232M-Q245S + A048G +T022Q-S099G-S101A-S103N-V104I-A232V-Q245R +S024R-R045T-S101A-G211Q-T213A +S024R-R045T-S101A-I107V-A114T-N116L-P210I-G211Q-T213A +T022R-S101T-S103G-V104I-A232L-Q245S + S099G-S103A + A098F-S099A +T022R-S101N-S103G-V104I-A232V-Q245W +T022A-S103A-V104I-L124T-A232V-Q245R +T022A-S024F-S101G-S103A-V104I-N116L-Y209V-A232V +T022K-S101N-S103N-V104L-A232V-Q245S +T022K-S101A-S103A-V104L-A232M-Q245R +T022Q-S101G-S103N-V104I-A232T-Q245W +T022A-S101T-S103A-V104L-A232T-Q245W +T022A-S101N-S103N-V104I-A232V-Q245S +T022A-N076D-S078N-G097A-S101N-S103A-V104I-L217Q-A232V- + Q245R A098M +T022A-S101G-S103G-V104I-A232L-Q245R + G020R-S101A-S207G-P210I-T213A +T022A-S024G-G097A-S101Q-S103A-V104I-Q109G-S128A-A232V- + Q245RG020R-S024R-S078G-G118S-G159D-Q245R-N248D-H249R +G020R-S024R-S078D-G118S-P129E-G159D-Q245R +T022Y-S101G-S103N-V104I-A232L-Q245S + V026Q + S240G +G020R-S024R-S078G-G118S-S188D + G020R-S024R-S078G-G118D-N238D-N248D +T022R-S101G-S103A-V104I-A232T-Q245S +T022W-S024R-R045T-S101A-P210I-G211Q +T022A-S024G-N076D-S078N-G097S-S101Q-S103A-V104I-A232V- + Q245RT022W-S024R-R045T-S101A-T213A + A200F +T022R-S101T-S103G-V104L-A232T-Q245S +T022A-S078N-G097S-S101Q-S103A-V104I-Q109N-L217Q-A232V- + Q245RT022A-S101G-S103N-V104I-A232T-Q245S +T022A-S101T-S103N-V104L-A232T-Q245S +T022A-S101A-S103A-V104L-A232V-Q245S + S099G-S101A-V104L +H017R-T022A-S024V-N076D-G097S-S101N-S103A-V104I-Q109N- +S128A-A232V-Q245R T022A-S101A-S103N-V104L-A232M-Q245S +T022A-S101Q-S103A-V104I-Q109G-L217Q-A232V-Q245R +T022A-S024G-N076D-S101Q-S103A-V104I-S128A-A232V-Q245R +T022A-S024G-S078N-G097S-S101N-S103A-V104I-S128A-S212P- +L217Q-A232V-Q245R S056L + T253L + T022W-R045T-S101A-G211Q-T213A +T022A-G097A-S101Q-S103A-V104I-S128A-L217Q-A232V-Q245R +T022A-S024G-N076D-S078N-G097A-S101N-S103A-V104I-S128A- + A232V-Q245RG020R-T022W-R045T-S101A-G211Q-T213A + S101A-G211Q + N123D +T022R-S101A-S103N-V104I-A232M-Q245W +T022Y-S101T-S103N-V104L-A232T-Q245R +T022A-S101G-S103A-V104I-N116L-Y209V-G211Q-T213A-A232V +G020R-S024R-S078D-G118D-N248D + A098Q-S099G-S101A + S099T-V104I +T022Q-S101G-S103A-V104I-A232V-Q245S +T022R-S101A-S103G-V104I-A232T-Q245S +T022K-S101T-S103N-V104I-A232T-Q245W + A016M +T022A-S024G-N076D-S101N-S103A-V104I-Q109G-A232V-Q245R +T022K-S101N-S103N-V104I-A232T-Q245S +G020R-N043R-R045T-S101A-N204D-P210I-G211Q +T022A-S101T-S103N-V104I-A232V-Q245S +T022Y-S101G-S103N-V104L-A232V-Q245R +T022A-S101T-S103N-V104I-A232V-Q245W +T022A-S024G-S078N-G097S-S101N-S103A-V104I-Q109G-L217Q- + A232V-Q245RA088T + T022Y-S101A-S103A-V104I-A232M-Q245S +T022W-S024R-R045T-S101A-N116L-P210I-G211Q +T022W-N043R-R045T-S101A-P210I-G211Q-T213A + T038A +S078D-G118S-G159D-Q245R + T022K-S101T-S103A-V104L-A232V-Q245W +T022A-S024G-N076D-S078N-G097S-S101N-S103A-V104I-Q109G- + A232V-Q245RT022A-S078N-S101Q-S103A-V104I-Q109N-L217Q-A232V-Q245R + G025S + G025T +A016S-S101G-S103A-V104I-N116L-M119V-Y209V-G211Q-A232V +T022A-N076D-V084A-S101G-S103A-V104I-A232V-Q245R + V011L +T022Y-S101T-S103G-V104I-A232M-Q245S +T022R-S101N-S103A-V104L-A232T-Q245S + S240V +G020R-S024R-S078G-G118D-P129E-S188D-H249R +T022W-S024R-R045T-S101A-G211Q-T213A +T022A-S024G-N076D-G097A-S101Q-S103A-V104I-Q109G-A232V- + Q245R V004Y +V051A + G195W-N269E + S024R-S078G-G118D-N248D-H249R +T022A-S099G-S101T-S103G-V104I-A232V-Q245S +T022Q-S101T-S103G-V104I-A232L-Q245W +T022Q-S101N-S103N-V104I-A232V-Q245W +T022A-S101T-S103G-V104I-A232V-Q245W +T022K-S101N-S103G-V104I-A232V-Q245R +T022Q-S101G-S103N-V104I-A232V-Q245S +

TABLE 2-5 BMI cleaning performance of Group C2 variants. PI Relative toGG36; Sequence of GG36 Variants (BPN′ BMI assay, Detergent numbering)107 at 25° C. G020R-S024R-S078N-G097A-S101A-Q109N-N116L-L217Q +++T022R-S024F-S101G-S103A-V104I-A232V-Q245R-E271H +++G020R-S024R-S078N-G097S-S101Q-S128A-L217Q +++T022R-S101G-S103A-V104I-A232V-Q245R-E271H +++G020R-S024R-S101G-N116L-S128A +++ G020R-S024R-S101A-Q109N-N116L-S128A+++ T022R-S024F-S101G-S103A-V104I-A232V-Q245R-T260M +++G020R-S024R-S078N-S101N-N116L-S128A +++G020R-S024R-G097A-S101A-Q109G-N116L +++ G020R-S024R-S101N-S128A +++G020R-S024R-S078N-S101G-S128A-L217Q +++T022R-S024F-S101G-S103A-V104I-A232V-Q245R +++T022R-S078N-G097S-S101Q-S103A-V104I-Q109N-N116L-A232V- +++ Q245RG020R-S024R-G097A-S101A-Q109N-N116L-S128A +++G020R-S024R-G097S-S101N-Q109N-S128A +++T022R-S101G-S103A-V104I-A232V-P239G-Q245R +++G020R-S024R-G097S-S101A-S128A +++ G020R-S024R-S101G-Q109N-N116L-A215V+++ G020R-S024R-S078N-G097S-S101Q-Q109G-N116L-S128A +++G020R-S024R-S078N-G097S-S101N-Q109N +++ G020R-S024R-S101G-S128A +++T022R-S101G-S103A-V104I-A232V-Q245R-T260K-L267N +++T022R-S101G-S103A-V104I-S128A-A232V-Q245R +++T022R-S024G-G097S-S101N-S103A-V104I-A232V-Q245R +++T022R-S024F-S101G-S103A-V104I-V121F-A232V-Q245R +++G020R-S024R-G097A-S101Q-N116L-S128A-L217Q +++T022R-S101G-S103A-V104I-A232V-Q245R +++T022R-G097A-S101N-S103A-V104I-N116L-A232V-Q245R +++G020R-S024R-S078N-G097S-S101A-N116L-S128A +++G020R-S024R-G097A-S101G-S128A-L217Q +++T022R-S078N-S101G-S103A-V104I-N116L-A232V-Q245R +++G020R-S024G-N043R-S078N-S101Q-Q109G-N116L-S128A-Q206R- +++ L217QG020R-S024R-S078N-G097S-S101Q-N116L-S128A +++G020R-S024R-S078N-G097S-S101N-Q109N-N116L-S128A +++G020R-S024R-S101N-Q109G-N116L +++ G020R-S024R-S101Q-N116L-S128A-L217Q+++ G020R-N043R-S078N-S101G-Q109G-N116L +++G020R-S024G-N043R-S078N-G097S-S101G-S128A +++T022R-S024G-S078N-S101N-S103A-V104I-S128A-A232V-Q245R +++T022R-S024G-G097A-S101Q-S103A-V104I-Q109G-N116L-S128A- +++ A232V-Q245RT022R-S101G-S103A-V104I-A232V-P239G-Q245R-E271I +++G020R-S024R-S101Q-S128A +++ G020R-S024R-G097A-S101N-Q109G-N116L +++G020R-S024G-N043R-S078N-G097A-S101G-Q109G-N116L +++G020R-S024R-G097S-S101Q-Q109N-N116L-L217Q +++T022R-S024G-S078N-G097A-S101Q-S103A-V104I-A232V-Q245R +++G020R-S024R-G097A-S101G-Q109N-S128A +++G020R-S024R-G097S-S101A-Q109N-S128A +++T022R-G097S-S101Q-S103A-V104I-Q109G-A232V-Q245R +++G020R-S024R-S078N-G097A-S101N-S128A-L217Q +++G020R-S024G-N043R-S078N-G097S-S101G-N116L +++G020R-S024R-S078N-G097S-S101G-Q109N-N116L-L217Q +++T022R-S024G-S078N-S101N-S103A-V104I-Q109N-N116L-S128A- +++ A232V-Q245RG020R-S024R-S078N-G097S-S101A-Q109N-S128A-L217Q +++G020R-S024R-S078N-G097A-S101G-Q109N-N116L-L217Q +++G020R-S024G-N043R-S078N-S101Q +++G020R-S024R-S078N-G097A-S101Q-Q109G-N116L +++G020R-N043R-G097A-S101N-N116L-S128A +++G020R-S024R-S078N-S101G-Q109G-N116L-S128A-L217Q +++G020R-S024R-S078N-S101Q-Q109G-N116L-S128A-L217Q +++G020R-S024R-S078N-G097S-S101G-Q109N-N116L-S128A +++G020R-S024R-S078N-G097S-S101N-Q109N-N116L +++T022R-S101N-S103A-V104I-Q109N-N116L-S128A-A232V-Q245R +++T022R-S024G-G097S-S101G-S103A-V104I-Q109G-N116L-S128A- +++ A232V-Q245RT022R-S024G-S078N-S101G-S103A-V104I-N116L-S128A-A232V- +++ Q245RG020R-S024R-S078N-S101N-Q109N-N116L +++G020R-S024R-S078N-S101A-Q109N-N116L-S128A +++T022R-S024G-G097A-S101Q-S103A-V104I-N116L-S128A-A232V- +++ Q245RT022R-G097S-S101G-S103A-V104I-S128A-A232V-Q245R +++T022R-G097A-S101Q-S103A-V104I-S128A-A232V-Q245R +++T022R-S024G-S078N-G097S-S101Q-S103A-V104I-S128A-A232V- +++ Q245RT022R-S078N-G097A-S101Q-S103A-V104I-N116L-S128A-A232V- +++ Q245RG020R-S024R-S101G-Q109G-N116L-L217Q +++G020R-S024R-S078N-S101Q-Q109N-N116L +++G020R-S024R-S101G-N116L-S128A-L217Q +++G020R-S024R-S101A-Q109G-N116L-S128A +++T022R-S024G-S078N-G097A-S101G-S103A-V104I-N116L-L217Q- +++ A232V-Q245RT022R-S024G-S078N-G097S-S101Q-S103A-V104I-Q109G-N116L- +++S128A-A232V-Q245R G020R-N043R-G097A-S101N-S128A-L233I +++T022R-S101Q-S103A-V104I-A232V-Q245R +++G020R-N043R-G097S-S101Q-Q109G-N116L-S128A +++T022R-S078N-S101Q-S103A-V104I-A232V-Q245R +++G020R-S024R-G097S-S101N-Q109G-N116L +++T022R-S078N-G097S-S101G-S103A-V104I-S128A-A232V-Q245R +++T022R-S024G-S101N-S103A-V104I-A232V-Q245R +++G020R-S024R-G097A-S101N-Q109G-S128A +++G020R-N043R-S101Q-N116L-S128A-L217Q +++G020R-S024R-S078N-G097A-S101Q-Q109N-S128A +++G020R-S024R-E089D-S101A-Q109N-N116L +++G020R-S024R-G097A-S101G-Q109G-S128A +++G020R-S024G-N043R-S101G-N116L-S128A-L217Q +++G020R-N043R-S078N-S101Q-Q109G-L217Q +++G020R-S024G-N043R-G097A-S101N-Q109N-N116L-L217Q +++G020R-S024R-S078N-G097A-S101G-N116L-S128A +++G020R-S024R-G097S-S101A-Q109N-N116L +++G020R-S024R-S078N-S101A-Q109G-S128A +++G020R-S024R-G097A-S101A-Q109G-N116L-S128A +++G020R-N043R-G097A-S101Q-N116L-S128A +++G020R-N043R-G097S-S101Q-S128A-L217Q +++G020R-S024R-S078N-G097A-S101G-Q109G-N116L-L217Q +++G020R-N043R-G097A-S101G-Q109N-N116L-S128A +++G020R-N043R-S078N-G097S-S101N-Q109N-L217Q +++G020R-S024R-G097S-A098E-S101A-N116L-S128A +++G020R-S024R-S078N-G097S-S101A-Q109G-S128A +++T022R-S024G-S078N-G097S-S101G-S103A-V104I-Q109N-N116L- +++L217Q-A232V-Q245R G020R-S024R-S078N-G097A-S101Q-Q109N-N116L-L217Q +++T022R-S024G-G097S-S101Q-S103A-V104I-S128A-A232V-Q245R +++G020R-S024R-S101A-Q109N-N116L-S128A-L217Q +++G020R-S024R-S101G-Q109G-N116L-S128A-L217Q +++T022R-S078N-G097A-S101N-S103A-V104I-S128A-A232V-Q245R +++G020R-S024G-N043R-S101N-N116L-S128A-L217Q +++G020R-S024R-S078N-G097S-S101A-Q109N-N116L +++G020R-S024R-S078N-G097A-S101N-Q109N-N116L-S128A +++G020R-S024R-S078N-S101Q-Q109N-N116L-L217Q +++T022R-S024G-S078N-S101N-S103A-V104I-N116L-S128A-A232V- +++ Q245RG020R-N043R-S101G-Q109N-N116L-S128A-L217Q +++T022R-S101N-S103A-V104I-L217Q-A232V-Q245R +++T022R-G097A-S101N-S103A-V104I-N116L-S128A-A232V-Q245R +++G020R-S024R-G097A-S101A-S128A-L217Q +++T022R-S024G-S078N-G097A-S101N-S103A-V104I-S128A-L217Q- +++ A232V-Q245RG020R-S024R-S078N-G097S-S101N-Q109G-N116L-S128A-L217Q +++T022R-S024G-S101G-S103A-V104I-N116L-L217Q-A232V-Q245R +++T022R-G097A-S101Q-S103A-V104I-N116L-S128A-A232V-Q245R +++G020R-N043R-S101Q-S128A-L217Q +++G020R-S024R-S078N-S101N-Q109G-S128A-L217Q +++G020R-S024R-S078N-G097A-S101N-Q109N-N116L-S128A-L217Q +++T022R-S024G-G097A-S101Q-S103A-V104I-N116L-L217Q-A232V- +++ Q245RG020R-S024R-S078N-G097S-S101G-S128A-N204S-L217Q +++T022R-S024G-S078N-S101Q-S103A-V104I-N116L-S128A-A232V- +++ Q245RG020R-S024R-S078N-G097S-S101Q-Q109N-N116L-L217Q +++G020R-S024R-S078N-S099G-S101Q-Q109N-N116L-L217Q +++T022R-S024G-G097A-S101G-S103A-V104I-Q109G-N116L-L217Q- +++ A232V-Q245RT022R-S078N-G097A-S101Q-S103A-V104I-S128A-L217Q-A232V- +++ Q245RT022R-G097A-S101G-S103A-V104I-Q109G-N116L-L217Q-A232V- +++ Q245RG020R-S024R-G097A-S101N-Q109N-S128A-L217Q +++T022R-G097S-S101N-S103A-V104I-S128A-L217Q-A232V-Q245R +++G020R-S024R-K094R-S101N-N116L-S128A-L217Q +++T022R-S024G-S078N-G097A-S101G-S103A-V104I-N116L-S128A- +++L217Q-A232V-Q245R G020R-S024R-G097A-S101A-N116L-S128A-L217Q +++G020R-S024R-S078N-G097W-S101Q-Q109N-N116L-L217Q +++T022R-G097S-S101N-S103A-V104I-N116L-S128A-L217Q-A232V- +++ Q245RT022R-S101Q-S103A-V104I-N116L-L217Q-A232V-Q245R +++T022R-S024G-S078N-G097A-S101Q-S103A-V104I-N116L-S128A- +++L217Q-A232V-Q245R T022R-S078N-G097A-S101Q-S103A-V104I-Q109N-N116L-S128A-+++ L217Q-A232V-Q245R G020R-N043R-G097S-S101Q-Q109G-N116L-S128A-L217Q+++ T022R-S024G-G097A-S101G-S103A-V104I-N116L-S128A-L217Q- +++A232V-Q245R T022R-G097S-S101G-S103A-V104I-L217Q-A232V-Q245R +++T022R-S024G-G097A-S101Q-S103A-V104I-Q109G-N116L-L217Q- +++ A232V-Q245RT022R-S024G-S078N-G097A-S101Q-S103A-V104I-S128A-L217Q- +++ A232V-Q245RT022R-S101Q-S103A-V104I-Q109G-N116L-S128A-L217Q-A232V- +++ Q245RT022R-S078N-G097S-S101G-S103A-V104I-N116L-S128A-L217Q- +++ A232V-Q245RT022R-S024G-T071A-S078N-S101N-S103A-V104I-Q109N-N116L- +++S128A-A232V-Q245R T022R-S024G-G097A-S101Q-S103A-V104I-S128A-L217Q-A232V-+++ Q245R T022R-S101G-S103A-V104I-Q109N-S128A-A232V-Q245R +++T022R-S024G-G097S-S101G-S103A-V104I-S128A-L217Q-A232V- +++ Q245RT022R-S024G-S078N-S101Q-S103A-V104I-Q109N-N116L-S128A- +++L217Q-A232V-Q245R T022R-G097S-S101Q-S103A-V104I-S128A-L217Q-A232V-Q245R++ T022R-G097A-S101Q-S103A-V104I-Q109N-S128A-A232V-Q245R ++R010A-T022R-S024F-S101G-S103A-V104I-A232V-Q245R-L267N- ++ E271HG020R-S024G-N043R-S078N-S101G-Q109N-N116L-L217Q-M222S ++T022R-S078N-G097S-S101G-S103A-V104I-Q109G-S128A-A232V- + Q245RT022R-S101G-S103A-V104I-M222S-A232V-Q245R-E271H +G020R-S024R-S101N-Q109G-N116L-M222S + PI = Performance Index PI > or =1.50 is +++; PI between 1.49 and 1.20 = ++; PI between 1.19 and 1.1 = +in Detergent Composition 107 as described in Table 1

TABLE 2-6 BMI cleaning performance of Group C3 variants. PI Relative toGG36; Sequence of GG36 Variants (BPN′ BMI assay, Detergent numbering)107 at 25° C. T022R-S101G-S103A-A232V-Q245R +++T022R-S101G-S103A-V104I-A232V- +++ Q245R-E271AT022R-S103A-V104I-A232V-Q245R +++ T022R-S101G-V104I-A232V-Q245R +++T022R-S101G-S103A-V104I-Q245R +++ G020R-N043R-E271A +++G020R-S024R-I035T-S101A-N116L +++ G020R-N043R-P239G +++G020R-N043R-S242L +++ G020R-N043R-V234F +++ G020R-N043R-E271H ++G020R-N043R-E271I ++ G020R-S024R-S101A-N116L-N269I ++T022R-S101G-S103A-V104I-A232V- ++ Q245R-E271F G020R-N043R-E271F ++G020R-N043R-N269I ++ G020R-S024R-S101A-N116L-E271H ++T022R-S101G-S103A-V1041-A232V- ++ Q245R-E271H G020R-I035T-N043R ++G020R-S024R-S101A-A114T-N116L ++ G020R-S024R-S101A-N116L-E2711 ++G020R-N043R-A114T ++ S101G-S103A-V104I-A232V-Q245R ++ G020R-N043R-L267N++ G020R-S024R-S101A-N116L-L267N ++ G020R-S024R-V028A-S101A-N116L ++G020R-S024R-S101A-N116L-G258R ++ G020R-N043R-G258R ++G020R-S024R-S101A-N116L-S242L ++ G020R-S024R-S101A-N116L-P239G ++I008N-G020R-S024R-S101A-N116L ++ G020R-S024R-S101A-N116L-V234F ++T022R-S101G-S103A-V104I-A232V ++ G020R-S024R-S101A-N116L-E271A ++R010A-G020R-N043R ++ G020R-S024R-S101A-N116L-E271F ++ G020R-V028A-N043R++ I008N-G020R-N043R ++ G020R-S024F-S101A-N116L ++R010A-G020R-S024R-S101A-N116L + G020R-N043R-M222S + PI = PerformanceIndex PI > or = 1.50 is +++; PI between 1.49 and 1.20 = ++; PI between1.19 and 1.1 = + in Detergent Composition 107

We claim:
 1. An isolated subtilisin variant, wherein said subtilisinvariant is a mature form having proteolytic activity and comprises anamino acid sequence comprising a combination of amino acid substitutionsat positions selected from T022R+S101G+Q245R, wherein said subtilisinvariant has at least 80% amino acid identity with a Bacillus lentussubtilisin GG36 protease comprising the amino acid sequence shown in SEQID NO:2, and wherein amino acid positions of the subtilisin variant arenumbered according to the numbering of corresponding amino acidpositions in the amino acid sequence of Bacillus amyloliquefacienssubtilisin BPN′ shown in SEQ ID NO:1.
 2. The isolated subtilisin variantof claim 1, wherein the subtilisin variant is a subtilisin variant ofBacillus lentus subtilisin GG36 protease, wherein said Bacillus lentussubtilisin GG36 protease comprises the amino acid sequence shown in SEQID NO:2.
 3. The isolated subtilisin variant of claim 1, wherein thetotal net charge of the variant is 0, +1, +2, +3, +4, +5, −1, −2, −3,−4, or −5 relative to the total net charge of the Bacillus lentussubtilisin GG36 protease.
 4. The isolated subtilisin variant of claim 3,wherein the total net charge is obtained by one or more substitutionsselected from: A001E, V004E, R010H/A, Q012E, A015D, N018D, R019H/S,V026D, K027E, N043D, R045C/T/S/P, S049D, V051E, G061E, N062D/E, N076D,N077D, S078D, S087D, A098E, S101D, S106E, G115E, G118D, N123D, S128D,P129E, S130D/E, S132D, A158E, G159D/E, S160D, S166D, R170T, N183D,N184D, N185E, R186H, S188D/E, A194E, A200D, Y209E, A215D, N204D, S212D,L217D/E, N218D, A230E, K235F, K237E, N238D, S240D, N243D, Q245D, R247L,N248D/E, K251C, N263D, N269D/E, A272D, A273E, or R275H/S, and whereinamino acid positions of the protease variant are numbered according tothe numbering of corresponding amino acid positions in the amino acidsequence of Bacillus amyloliquefaciens subtilisin BPN′ shown in SEQ IDNO:1.
 5. The isolated subtilisin variant of claim 3, wherein the totalnet charge is obtained by one or more substitutions selected from:A001R, V004R, Q012R, P014R, H017R, N018R/K, G020K/R, T022R/K, S024R/K,G025R, D032I, D041K/L/N, N043R/K, G046R, A048R, F050R/K, P055R, S056R/K,T057R, Q059K, G061R, N076K, S078R, P086R, S087R, E089G/P/I, G097R,S099R, Q109R, G115R, G118R, S132K, S144R, G159R/K, D181C/S/T, L196K,N204K, Q206R, K235R, Q236R/K, K237R, N238R, S240R, W241R, S242R/K,V244R, Q245R/K, N248R, H249R, N252R/K, S256R, G258R, T260K, N269R/K, orE271F/H/T/L/W/R/S/I/A/G/V, and wherein amino acid positions of theprotease variant are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence ofBacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.
 6. Theisolated subtilisin variant of claim 1, wherein the subtilisin varianthas one or more of the following characteristics: a) a Test Method 2performance index of at least 1.1, at least 1.2, at least 1.3, at least1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least1.9, at least 2, from 1.1 to about 10, from 1.1 to about 8, or even from1.1 to about 5; b) a Test Method 3 performance index of at least 1.1, atleast 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, atleast 1.7, at least 1.8, at least 1.9, at least 2, from 1.1 to about 10,from 1.1 to about 8, or even from 1.1 to about 5; c) a Test Method 4performance index of at least 1.0, at least 1.1, at least 1.2, at least1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least1.8, at least 1.9, at least 2, from 1.0 to about 10, from 1.0 to about8, or even from 1.0 to about 5; and/or d) a Test Method 6 performanceindex of at least 1.0, at least 1.1, at least 1.2, at least 1.3, atleast 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, atleast 1.9, at least 2, from 1.0 to about 10, from 1.0 to about 8, oreven from 1.0 to about 5; and/or e) Test Method 7 performance index ofat least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, atleast 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from1.1 to about 15, from 1.1 to about 10, or even from 1.1 to about
 7. 7. Acomposition comprising at least one subtilisin variant of claim 1,wherein said composition is not a fabric and home care product.
 8. Thecomposition of claim 7, wherein said composition is a cleaningcomposition.
 9. The composition of claim 8, wherein said cleaningcomposition is a granular, powder, solid, bar, liquid, tablet, gel, orpaste composition.
 10. The composition of claim 7, further comprising atleast one bleaching agent.
 11. The composition of claim 7, wherein saidcleaning composition is phosphate-free.
 12. The composition of claim 7,wherein said cleaning composition contains phosphate.
 13. Thecomposition of claim 7, further comprising at least one additionalenzyme.
 14. The composition of claim 13, wherein said at least oneadditional enzyme is selected from hemicellulases, cellulases,peroxidases, proteases, metalloproteases, xylanases, lipases,phospholipases, esterases, perhydrolases, cutinases, pectinases, pectatelyases, mannanases, keratinases, reductases, oxidases, phenoloxidases,lipoxygenases, ligninases, pullulanases, tannases, pentosanases,malanases, β-glucanases, arabinosidases, hyaluronidases,chondroitinases, laccases, and amylases, or any combination thereof. 15.The composition of claim 7, wherein said subtilisin variant is not acold water protease variant, wherein said cold water protease varianthas one or more of the following characteristics: a) a Test Method 2performance index of at least 1.1, at least 1.2, at least 1.3, at least1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least1.9, at least 2, from 1.1 to about 10, from 1.1 to about 8, or even from1.1 to about 5; b) a Test Method 3 performance index of at least 1.1, atleast 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, atleast 1.7, at least 1.8, at least 1.9, at least 2, from 1.1 to about 10,from 1.1 to about 8, or even from 1.1 to about 5; c) a Test Method 4performance index of at least 1.0, at least 1.1, at least 1.2, at least1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least1.8, at least 1.9, at least 2, from 1.0 to about 10, from 1.0 to about8, or even from 1.0 to about 5; and/or d) a Test Method 6 performanceindex of at least 1.0, at least 1.1, at least 1.2, at least 1.3, atleast 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, atleast 1.9, at least 2, from 1.0 to about 10, from 1.0 to about 8, oreven from 1.0 to about 5; and/or e) Test Method 7 performance index ofat least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, atleast 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from1.1 to about 15, from 1.1 to about 10, or even from 1.1 to about
 7. 16.A method of cleaning, comprising contacting a surface or an item with acleaning composition comprising at least one subtilisin variant ofclaim
 1. 17. A method of cleaning comprising contacting a surface or anitem with a cleaning composition set forth in claim
 7. 18. The method ofclaim 16, wherein said subtilisin variant is not a cold water proteasevariant, wherein said cold water protease variant has one or more of thefollowing characteristics: a) a Test Method 2 performance index of atleast 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, atleast 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from1.1 to about 10, from 1.1 to about 8, or even from 1.1 to about 5; b) aTest Method 3 performance index of at least 1.1, at least 1.2, at least1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least1.8, at least 1.9, at least 2, from 1.1 to about 10, from 1.1 to about8, or even from 1.1 to about 5; c) a Test Method 4 performance index ofat least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, atleast 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, atleast 2, from 1.0 to about 10, from 1.0 to about 8, or even from 1.0 toabout 5; and/or d) a Test Method 6 performance index of at least 1.0, atleast 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, atleast 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from1.0 to about 10, from 1.0 to about 8, or even from 1.0 to about 5;and/or e) Test Method 7 performance index of at least 1.1, at least 1.2,at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, atleast 1.8, at least 1.9, at least 2, from 1.1 to about 15, from 1.1 toabout 10, or even from 1.1 to about
 7. 19. The isolated subtilisinvariant of claim 1, wherein said variant is selected fromT022R-S101G-S103A-V104I-A232V-Q245R;T022R-S024F-S101G-S103A-V104I-A232V-Q245R-E271H;T022R-S101G-S103A-V104I-A232V-Q245R-E271H;T022R-S024F-S101G-S103A-V104I-A232V-Q245R-T260M;T022R-S024F-S101G-S103A-V104I-A232V-Q245R;T022R-S101G-S103A-V104I-A232V-P239G-Q245R;T022R-S101G-S103A-V104I-A232V-Q245R-T260K-L267N;T022R-S101G-S103A-V104I-S128A-A232V-Q245R;T022R-S024F-S101G-S103A-V104I-V121F-A232V-Q245R;T022R-S078N-S101G-S103A-V104I-N116L-A232V-Q245R;T022R-S101G-S103A-V104I-A232V-P239G-Q245R-E271I;T022R-S024G-G097S-S101G-S103A-V104I-Q109G-N116L-S128A-A232V-Q245R;T022R-S024G-S078N-S101G-S103A-V104I-N116L-S128A-A232V-Q245R;T022R-G097S-S101G-S103A-V104I-S128A-A232V-Q245R;T022R-S024G-S078N-G097A-S101G-S103A-V104I-N116L-L217Q-A232V-Q245R;T022R-S024G-S078N-G097S-S101G-S103A-V104I-Q109N-N116L-L217Q-A232V-Q245R;T022R-S024G-S101G-S103A-V104I-N116L-L217Q-A232V-Q245R;T022R-S024G-G097A-S101G-S103A-V104I-Q109G-N116L-L217Q-A232V-Q245R;T022R-G097A-S101G-S103A-V104I-Q109G-N116L-L217Q-A232V-Q245R;T022R-S024G-S078N-G097A-S101G-S103A-V104I-N116L-S128A-L217Q-A232V-Q245R;T022R-S024G-G097A-S101G-S103A-V104I-N116L-S128A-L217Q-A232V-Q245R;T022R-G097S-S101G-S103A-V104I-L217Q-A232V-Q245R;T022R-S078N-G097S-S101G-S103A-V104I-N116L-S128A-L217Q-A232V-Q245R;T022R-S101G-S103A-V104I-Q109N-S128A-A232V-Q245R;T022R-S024G-G097S-S101G-S103A-V104I-S128A-L217Q-A232V-Q245R;T022R-S101G-S103A-V104I-A232V-Q245R-E271A;R010A-T022R-S024F-S101G-S103A-V104I-A232V-Q245R-L267N-E271H;T022R-S101G-S103A-V104I-A232V-Q245R-E271F;T022R-S101G-S103A-V104I-A232V-Q245R-E271H;T022R-S078N-G097S-S101G-S103A-V104I-Q109G-S128A-A232V-Q245R; andT022R-S101G-S103A-V104I-M222S-A232V-Q245R-E271H.